Single-tube Ptprc SNP genotyping of JAXBoy (CD45.1) and C57BL/6J (CD45.2) mice by endpoint PCR and gel electrophoresis.

Allelic variation at the Ptprc gene, which encodes the pan-leukocyte marker CD45/Ly5, is commonly exploited to track hematopoietic reconstitution by flow cytometry in mixed bone marrow chimera transplant experiments. Historically, this was accomplished using bone marrow from C57BL/6 (Ptprcb/CD45.2/Ly5.2) and congenic B6.SJL-PtprcaPepcb/Boy (Ptprca/CD45.1/Ly5.1) mice. Recently, the Jackson Laboratory directly CRISPR-engineered the Ptprca allele in C57BL/6J mice. This new isogenic strain, termed JAXBoy, differs from wild-type C57BL/6J mice by two nucleotides, compared to the biologically significant 37 megabase (Mb) SJL interval retained in B6.SJL-PtprcaPepcb/Boy/J mice. Currently, Ptprc/CD45 variants are identified by flow cytometry or allele-specific real-time PCR, both of which require specialized workflows and equipment compared to standard genotyping of endpoint PCR products by gel electrophoresis. Here, we employed allele-specific oligonucleotides in conjunction with differential incorporation of a long non-specific oligo 5'-tail to allow for simultaneous identification of the Ptprca and Ptprcb alleles using endpoint PCR and gel electrophoresis. This method allows for integration of Ptprc genotyping into standard genotyping workflows, which use a single set of thermocycling and gel electrophoresis conditions. Importantly, the strategy of primer placement and tail addition described here can be adapted to discriminate similar single- or multi-nucleotide polymorphisms at other genomic loci.

Research and Analysis of Molecules such as CD28, CD45RA, CD45RO, CD38, HLA-DR, and CD57 on T Cells in Multiple Myeloma.

BACKGROUND: For many years it has been postulated that the immune system controls the progress of multiple myeloma (MM). However, the phenotypes of T cells in MM remain to be elucidated. In this study, we compared the phenotypes of T cells, which were obtained from the peripheral blood, in MM patients with those in healthy donors (HD). The expression of CCR7, CD57, CD28, HLA-DR, CD38, CD45RA, and CD45RO were assessed on T cells from MM patients and HDs using multicolor flow cytometry (MFC). METHODS: For this study, 17 newly diagnosed MM patients were selected, and 20 healthy people were selected as a control group. MFC was used to detect the markers on T cells. RESULTS: We detected significant increases in the expression levels of HLA-DR, CD38, and CD57on CD8+ T cells, significant decreases in the expression levels of CD28 and CD45RA on CD8+ T cells, and a decrease of CD4+ effec-tor T cells in MM patients, compared to the HD group. CONCLUSIONS: Our study shows that the accumulation of peripheral CD8+CD57+T cells, CD8+CD38high T cells, and CD8+HLA-DR+CD38high T cells is reflective of an ongoing antitumor T cell response and a progressive immune dysfunction in MM. During chemotherapy, the recovery of immune function can be monitored by detecting the proportion of activated molecules of T lymphocytes.

Tumor-associated CD8+T cell tolerance induced by erythroid progenitor cells.

Introduction: CD8+T cell tolerance plays an important role in tumor escape. Recent studies have shown that CD45+ erythroid progenitor cells (CD45+EPCs) generated through splenic extramedullary erythropoiesis suppress tumor immunity. However, the mechanism underlying how CD45+EPCs mediate CD8+T cell tolerance remains incompletely understood and requires further research. Methods: In this study, the antigen-processing abilities of CD45+EPCs was verified through both in vitro and in vivo experiments. We have used the method of co-culture in vitro and adoptive transfer experiments in vivo to explore the effects of CD45+EPCs on CD8+T cell tolerance. RNA-sequencing analysis and blocking experiments were used to evaluate the role of ROS in the CD45+EPC mediated tolerance of CD8+T cells. Finally, we incorporated uric acid into the adoptive transfer experiments to rescue the CD45+EPC mediated tumor-promoting effect. Results and discussion: We found that CD45+EPCs take up soluble proteins, present antigenic epitopes on their surface, and induce antigen-specific CD8+T cell anergy. In addition, we found that CD45+EPC directly nitrates tyrosine within the TCR/CD8 complex via the production of reactive oxygen species and peroxynitrite, preventing CD8+ T cells from responding to their specific peptide antigens. Furthermore, uric acid treatment effectively abolished the immunosuppressive effects of CD45+EPCs during CD8+T cell adoptive transfer, thereby enhancing the anti-tumor efficacy. These results demonstrated that CD8+T cell tolerance in tumor-bearing mice is induced by CD45+EPCs. The results of this study have direct implications for tumor immunotherapy.

TCRαß-depleted hematopoietic stem cell transplant and third-party CD45RA+ depleted adoptive cell therapy for treatment of post-transplant parvovirus B19 aplastic crisis.

This is a case report of a 6-year-old girl with relapsed B cell acute lymphoblastic leukemia in which adoptive cell therapy was applied successfully to treat refractory human parvovirus (HPV) B19 infection. Allogenic chimeric antigen receptor (CAR) T-cell therapy (bispecific CD19/CD22) was bridged to hematopoietic stem cell transplantation (HSCT) using a haploidentical paternal donor. However, HPV B19 DNAemia progressed and transfusion-related graft versus host disease occurred. After finding a third-party related donor with a better HLA match, haploidentical HPV B19-seropositive CD45RA+ depleted cells (16.5 × 106/kg) were administered and paternal TCRαß+ depleted stem cell were retransplanted. The HPV B19 DNAemia became negative within 1 week and the reticulocyte, neutrophil, hemoglobin, and platelet counts gradually normalized. The patient remained stable during the 1-year outpatient follow-up period. Thus, our case report highlights that persistent B19 infection can lead to pancytopenia, aplastic crisis, and graft rejection and TCRαß+ depleted haplo-HSCT is an effective means of hematopoiesis recovery. CD45RO memory T-cell therapy is the key to treating and preventing the development of refractory severe HPV B19 infection.

Alloengraftment without significant toxicity or GVHD in CD45 antibody-drug conjugate-conditioned Fanconi anemia mice.

ABSTRACT: Fanconi anemia (FA) is an inherited DNA repair disorder characterized by bone marrow (BM) failure, developmental abnormalities, myelodysplasia, leukemia, and solid tumor predisposition. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a mainstay treatment, is limited by conditioning regimen-related toxicity and graft-versus-host disease (GVHD). Antibody-drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) can open marrow niches permitting donor stem cell alloengraftment. Here, we report that single dose anti-mouse CD45-targeted ADC (CD45-ADC) facilitated stable, multilineage chimerism in 3 distinct FA mouse models representing 90% of FA complementation groups. CD45-ADC profoundly depleted host stem cell enriched Lineage-Sca1+cKit+ cells within 48 hours. Fanca-/- recipients of minor-mismatched BM and single dose CD45-ADC had peripheral blood (PB) mean donor chimerism >90%; donor HSCs alloengraftment was verified in secondary recipients. In Fancc-/- and Fancg-/- recipients of fully allogeneic grafts, PB mean donor chimerism was 60% to 80% and 70% to 80%, respectively. The mean percent donor chimerism in BM and spleen mirrored PB results. CD45-ADC-conditioned mice did not have clinical toxicity. A transient <2.5-fold increase in hepatocellular enzymes and mild-to-moderate histopathological changes were seen. Under GVHD allo-HSCT conditions, wild-type and Fanca-/- recipients of CD45-ADC had markedly reduced GVHD lethality compared with lethal irradiation. Moreover, single dose anti-human CD45-ADC given to rhesus macaque nonhuman primates on days -6 or -10 was at least as myeloablative as lethal irradiation. These data suggest that CD45-ADC can potently promote donor alloengraftment and hematopoiesis without significant toxicity or severe GVHD, as seen with lethal irradiation, providing strong support for clinical trial considerations in highly vulnerable patients with FA.

Histoplasty Modification of the Tumor Microenvironment in a Murine Preclinical Model of Breast Cancer.

PURPOSE: To develop a noninvasive therapeutic approach able to alter the biophysical organization and physiology of the extracellular matrix (ECM) in breast cancer. MATERIALS AND METHODS: In a 4T1 murine model of breast cancer, histoplasty treatment with a proprietary 700-kHz multielement therapy transducer using a coaxially aligned ultrasound (US) imaging probe was used to target the center of an ex vivo tumor and deliver subablative acoustic energy. Tumor collagen morphology was qualitatively evaluated before and after histoplasty with second harmonic generation. Separately, mice bearing bilateral 4T1 tumors (n = 4; total tumors = 8) were intravenously injected with liposomal doxorubicin. The right flank tumor was histoplasty-treated, and tumors were fluorescently imaged to detect doxorubicin uptake after histoplasty treatment. Next, 4T1 tumor-bearing mice were randomized into 2 treatment groups (sham vs histoplasty, n = 3 per group). Forty-eight hours after sham/histoplasty treatment, tumors were harvested and analyzed using flow cytometry. RESULTS: Histoplasty significantly increased (P = .002) liposomal doxorubicin diffusion into 4T1 tumors compared with untreated tumors (2.12- vs 1.66-fold increase over control). Flow cytometry on histoplasty-treated tumors (n = 3) demonstrated a significant increase in tumor macrophage frequency (42% of CD45 vs 33%; P = .022) and a significant decrease in myeloid-derived suppressive cell frequency (7.1% of CD45 vs 10.3%; P = .044). Histoplasty-treated tumors demonstrated increased CD8+ (5.1% of CD45 vs 3.1%; P = .117) and CD4+ (14.1% of CD45 vs 11.8%; P = .075) T-cell frequency. CONCLUSIONS: Histoplasty is a nonablative focused US approach to noninvasively modify the tumor ECM, increase chemotherapeutic uptake, and alter the tumor immune microenvironment.

Performance of a novel eight-color flow cytometry panel for measurable residual disease assessment of chronic lymphocytic leukemia.

BACKGROUND: Measurable residual disease (MRD) is an important prognostic indicator of chronic lymphocytic leukemia (CLL). Different flow cytometric panels have been developed for the MRD assessment of CLL in Western countries; however, the application of these panels in China remains largely unexplored. METHODS: Owing to the requirements for high accuracy, reproducibility, and comparability of MRD assessment in China, we investigated the performance of a flow cytometric approach (CD45-ROR1 panel) to assess MRD in patients with CLL. The European Research Initiative on CLL (ERIC) eight-color panel was used as the "gold standard." RESULTS: The sensitivity, specificity, and concordance rate of the CD45-ROR1 panel in the MRD assessment of CLL were 100% (87/87), 88.5% (23/26), and 97.3% (110/113), respectively. Two of the three inconsistent samples were further verified using next-generation sequencing. In addition, the MRD results obtained from the CD45-ROR1 panel were positively associated with the ERIC eight-color panel results for MRD assessment (R = 0.98, p < 0.0001). MRD detection at low levels (≤1.0%) demonstrated a smaller difference between the two methods (bias, -0.11; 95% CI, -0.90 to 0.68) than that at high levels (>1%). In the reproducibility assessment, the bias was smaller at three data points (within 24, 48, and 72 h) in the CD45-ROR1 panel than in the ERIC eight-color panel. Moreover, MRD levels detected using the CD45-ROR1 panel for the same samples from different laboratories showed a strong statistical correlation (R = 0.99, p < 0.0001) with trivial interlaboratory variation (bias, 0.135; 95% CI, -0.439 to 0.709). In addition, the positivity rate of MRD in the bone marrow samples was higher than that in the peripheral blood samples. CONCLUSIONS: Collectively, this study demonstrated that the CD45-ROR1 panel is a reliable method for MRD assessment of CLL with high sensitivity, reproducibility, and reliability.

Application of Blood Lymphocyte Immunophenotype and TCR Gene Rearrangement in the Diagnosis of CTCL.

BACKGROUND: The immunophenotype of peripheral blood lymphocytes and T-cell receptor (TCR) gene rearrangement of cutaneous T cell lymphoma (CTCL) patients were retrospectively analyzed to explore their value in the diagnosis of CTCL. METHODS: A total of fifty patients' results were enrolled from 2013 to 2021, including 29 malignant skin disorders and 21 benign skin disorders. The immunophenotype of peripheral blood lymphocytes were analyzed by flow cytometry and TCR gene rearrangement was detected by capillary electrophoresis. Lymphocyte subsets, CD4/CD8 ratio, the percentage of CD3+CD4+CD7- cells and CD45RA/CD45RO ratio was calculated between malignant and benign skin disorders. Peripheral blood lymphocyte immunophenotype and TCR gene rearrangement was compared with skin biopsy to evaluate their sensitivity and specificity. RESULTS: Lymphocyte subsets between malignant and benign groups have no significant difference in percentage of T cell (p > 0.05). The CD4/CD8 ratio is higher in patients with malignant lymphoma than the healthy range. The percentage of CD3+CD4+CD7- cells in malignant groups is higher than that in benign groups and CD45RA/ CD45RO ratio has significant difference between malignant and benign groups (p < 0.05). The sensitivity and specificity of TCR rearrangement for CTCL were 51.7% and 42.9%. The sensitivity and specificity of peripheral blood lymphocyte immunophenotype for CTCL were 44.8% and 33.3%. Combining the two methods, the sensitivity and specificity reached 69.0% and 38.1%, respectively. CONCLUSIONS: CD4/CD8 ratio of lymphocyte subsets, the proportion of CD4+CD7-T cells and CD45RA/CD45RO ratio can effectively distinguish benign and malignant dermatosis. TCR rearrangement method combined with lymphocyte immunophenotype can improve the sensitivity and specificity of CTCL diagnosis.

Prognostic Value of Immune Cells Subsets Within the Tumor Microenvironment in Patients With Rectal Adenocarcinoma.

BACKGROUND/AIM: One-third of newly diagnosed colorectal cancer cases are rectal cancers. Multimodal treatment regimens including surgery, radiotherapy, and chemotherapy improve local control and survival outcome and decrease tumor relapse for patients with rectal adenocarcinoma (READ). However, stratification of patients to predict their responses is urgently needed to improve therapeutic responses. PATIENTS AND METHODS: Immunostainings of CD3+, CD8+, and CD45RO+ immune cell subsets within the tumor microenvironment were evaluated using immunohistochemistry in two hundred seventy-nine READ patients. RESULTS: In this study, we found that examination of the adaptive immune response by quantifying CD3+, CD8+, and CD45RO+ immune cell subsets, provides improved and independent prognostic value for patients with READ. Regardless of conventional clinical and pathologic parameters, the densities of T cell subsets were strongly related to a better prognosis in patients with READ. High density of intratumoral immune cells is associated with absence of nodal metastasis, lymphovascular invasion, and perineural invasion. Moreover, high tumor-infiltrating lymphocyte (TIL) subsets were associated with favorable survival outcome in patients with READ, especially high-risk patients with advanced READ. CONCLUSION: Immune cell subsets including CD3, CD8, and CD45RO within the tumor microenvironment were independent prognostic factors for patients with READ.

Detection of effusion tumor cells under different storage and processing conditions.

BACKGROUND: Circulating tumor cells (CTCs) shed into blood provide prognostic and/or predictive information. Previously, the authors established an assay to detect carcinoma cells from pleural fluid, termed effusion tumor cells (ETCs), by employing an immunofluorescence-based CTC-identification platform (RareCyte) on air-dried unstained ThinPrep (TP) slides. To facilitate clinical integration, they evaluated different slide processing and storage conditions, hypothesizing that alternative comparable conditions for ETC detection exist. METHODS: The authors enumerated ETCs on RareCyte, using morphology and mean fluorescence intensity (MFI) cutoffs of >100 arbitrary units (a.u.) for epithelial cellular adhesion molecule (EpCAM) and <100 a.u. for CD45. They analyzed malignant pleural fluid from three patients under seven processing and/or staining conditions, three patients after short-term storage under three conditions, and seven samples following long-term storage at -80°C. MFI values of 4',6-diamidino-2-phenylindol, cytokeratin, CD45, and EpCAM were compared. RESULTS: ETCs were detected in all conditions. Among the different processing conditions tested, the ethanol-fixed, unstained TP was most similar to the previously established air-dried, unstained TP protocol. All smears and Pap-stained TPs had significantly different marker MFIs from the established condition. After short-term storage, the established condition showed comparable results, but ethanol-fixed and Pap-stained slides showed significant differences. ETCs were detectable after long-term storage at -80°C in comparable numbers to freshly prepared slides, but most marker MFIs were significantly different. CONCLUSIONS: It is possible to detect ETCs under different processing and storage conditions, lending promise to the application of this method in broader settings. Because of decreased immunofluorescence-signature distinctions between cells, morphology may need to play a larger role.

Combination of oligo-fractionated irradiation with nivolumab can induce immune modulation in gastric cancer.

BACKGROUND: Tumor-associated antigen (TAA)-specific CD8(+) T cells are essential for nivolumab therapy, and irradiation has been reported to have the potential to generate and activate TAA-specific CD8(+) T cells. However, mechanistic insights of T-cell response during combinatorial immunotherapy using radiotherapy and nivolumab are still largely unknown. METHODS: Twenty patients included in this study were registered in the CIRCUIT trial (ClinicalTrials.gov, NCT03453164). All patients had multiple distant metastases and were intolerance or had progressed after primary and secondary chemotherapy without any immune checkpoint inhibitor. In the CIRCUIT trial, eligible patients were treated with a total of 22.5 Gy/5 fractions/5 days of radiotherapy to the largest or symptomatic lesion prior to receiving nivolumab every 2 weeks. In these 20 patients, T-cell responses during the combinatorial immunotherapy were monitored longitudinally by high-dimensional flow cytometry-based, multiplexed major histocompatibility complex multimer analysis using a total of 46 TAAs and 10 virus epitopes, repertoire analysis of T-cell receptor ß-chain (TCRß), together with circulating tumor DNA analysis to evaluate tumor mutational burden (TMB). RESULTS: Although most TAA-specific CD8(+) T cells could be tracked longitudinally, several TAA-specific CD8(+) T cells were detected de novo after irradiation, but viral-specific CD8(+) T cells did not show obvious changes during treatment, indicating potential irradiation-driven antigen spreading. Irradiation was associated with phenotypical changes of TAA-specific CD8(+) T cells towards higher expression of killer cell lectin-like receptor subfamily G, member 1, human leukocyte antigen D-related antigen, T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain, CD160, and CD45RO together with lower expression of CD27 and CD127. Of importance, TAA-specific CD8(+) T cells in non-progressors frequently showed a phenotype of CD45RO(+)CD27(+)CD127(+) central memory T cells compared with those in progressors. TCRß clonality (inverted Pielou's evenness) increased and TCRß diversity (Pielou's evenness and Diversity Evenness score) decreased during treatment in progressors (p=0.029, p=0.029, p=0.012, respectively). TMB score was significantly lower in non-progressors after irradiation (p=0.023). CONCLUSION: Oligo-fractionated irradiation induces an immune-modulating effect with potential antigen spreading and the combination of radiotherapy and nivolumab may be effective in a subset of patients with gastric cancer.

Frequent expression of CD45RO memory T cell marker as well as low to high expression of PD-1 and PD-L1 inhibitory molecules in seminoma and dysgerminoma.

BACKGROUND: Seminoma and dysgerminoma are rare testicular and ovarian germ cell tumors characterized by a significant infiltration of immune cells in the tumor microenvironment. According to the failure of conventional treatments in some patients, it is crucial to identify novel prognostic and therapeutic biomarkers for these patients. The objectives of this study were to evaluate the expression of CD45RO and PD-1/PD-L1 and investigate their association with the clinicopathological characteristics of the patients. METHODS: Immunohistochemistry was performed to assess the expression of CD45RO, PD-1, and PD-L1 in tumor-infiltrated lymphocytes (TILs), and tumor cells in 33 seminoma and 31 dysgerminoma patients. The expression levels were evaluated using a semiquantitative approach, weighted histoscore, which considers both the intensity and extent of staining. RESULTS: All seminoma and dysgerminoma patients exhibited CD45RO expression in TILs, with 66.7 % and 90.3 % displaying high levels of expression, respectively. PD-1 expression in TILs was observed at low levels in 81.8 % and 77.4 % and at high levels in 18.2 % and 19.4 % of seminoma and dysgerminoma patients, respectively. Likewise, low expression of PD-L1 in tumor cells was detected in 63.6 % of seminoma and 61.3 % of dysgerminoma patients, while none of the patients exhibited high expression of PD-L1. In seminoma patients, a positive correlation was observed between PD-1 expression in TILs and CD45RO expression and between PD-L1 expression in tumor cells and TILs score. CONCLUSION: The frequent infiltration of CD45RO, along with variable expression of PD-1 and PD-L1 on TILs and tumor cells, could impact the effectiveness of anti-tumor responses and immunotherapy.

Generation of Non-Alloreactive Antiviral Central Memory CD8 Human Veto T Cells for Cell Therapy.

Previous studies in mice demonstrated that CD8 T cells exhibit marked veto activity enhancing engraftment in several models for T cell-depleted bone marrow (TDBM) allografting. To reduce the risk of graft-versus-host disease (GVHD) associated with allogeneic CD8 veto T cells, these studies made use of naive CD8 T cells stimulated against third-party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. More recently, it was shown that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen. These cells also exhibited substantial enhancement of BM allografting without GVHD. In this study, we tested the hypothesis that stimulation and expansion of human CD8 memory T cells under IL-15 and IL-7 deprivation during the early phase of activation against recall viral antigens can lead to substantial loss of alloreactive T clones while retaining marked veto activity. Memory CD8 T cells were enriched by removal of CD45RA+, CD4+, and CD56+ cells from peripheral blood of cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-positive donors. In parallel, CD14+ monocytes were isolated; differentiated into mature dendritic cells (mDCs); pulsed with a library of CMV, EBV, adenovirus, and BK virus peptides; and irradiated. The CD8 T cell-enriched fraction was then cultured with the pulsed mDCs in the presence of IL-21 for 3 days, after which IL-15 and IL-7 were added. After 12 days of culture, the cells were tested by limiting dilution analysis for the frequency of alloreactive T cell clones and their veto activity. In preclinical runs using GMP reagents, we established that within 12 days of culture, a large number of highly homogenous CD8 T cells, predominantly expressing a central memory phenotype, could be harvested. These cells exhibited marked veto activity in vitro and >3-log depletion of alloreactivity. Based on these preclinical data, a phase 1-2 clinical trial was initiated to test the safety and efficacy of these antiviral CD8 central memory veto cells in the context of nonmyeloablative (NMA) T cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT). In 2 validation runs and 11 clinical runs using GMP reagents, >1 × 1010 cells were generated from a single leukapheresis in 12 out of 13 experiments. At the end of 12 days of culture, there were 97 ± 2.5% CD3+CD8+ T cells, of which 84 ± 9.0% (range, 71.5% to 95.1%) exhibited the CD45RO+CD62L+ CM phenotype. Antiviral activity tested by intracellular expression of INF-γ and TNF-α and showed an average of 38.8 ± 19.6% positive cells on 6 hours of stimulation against the viral peptide mixture. Our results demonstrate a novel approach for depleting alloreactive T cell clones from preparations of antiviral CD8 veto cells. Based on these results, a phase 1-2 clinical trial is currently in progress to test the safety and efficacy of these veto cells in the context of NMA haploidentical T cell-depleted HSCT. Studies testing the hypothesis that these non-alloreactive CD8 T cells could potentially offer a platform for off-the-shelf veto chimeric antigen receptor T cell therapy in allogenic recipients, are warranted.

Product Attributes of CAR T-cell Therapy Differentially Associate with Efficacy and Toxicity in Second-line Large B-cell Lymphoma (ZUMA-7).

Treatment resistance and toxicities remain a risk following chimeric antigen receptor (CAR) T-cell therapy. Herein, we report pharmacokinetics, pharmacodynamics, and product and apheresis attributes associated with outcomes among patients with relapsed/refractory large B-cell lymphoma (LBCL) treated with axicabtagene ciloleucel (axi-cel) in ZUMA-7. Axi-cel peak expansion associated with clinical response and toxicity, but not response durability. In apheresis material and final product, a naive T-cell phenotype (CCR7+CD45RA+) expressing CD27 and CD28 associated with improved response durability, event-free survival, progression-free survival, and a lower number of prior therapies. This phenotype was not associated with high-grade cytokine release syndrome (CRS) or neurologic events. Higher baseline and postinfusion levels of serum inflammatory markers associated with differentiated/effector products, reduced efficacy, and increased CRS and neurologic events, thus suggesting targets for intervention. These data support better outcomes with earlier CAR T-cell intervention and may improve patient care by informing on predictive biomarkers and development of next-generation products. SIGNIFICANCE: In ZUMA-7, the largest randomized CAR T-cell trial in LBCL, a naive T-cell product phenotype (CCR7+CD45RA+) expressing CD27 and CD28 associated with improved efficacy, decreased toxicity, and a lower number of prior therapies, supporting earlier intervention with CAR T-cell therapy. In addition, targets for improvement of therapeutic index are proposed. This article is featured in Selected Articles from This Issue, p. 4.

Flow cytometric analysis of CD34+ CD38- cells; cell frequency and immunophenotype based on CD45RA expression pattern.

INTRODUCTION: The CD34+ CD38- population in bone marrow includes hematopoietic stem/progenitor cells. Recently, in acute myeloid leukemia, the focus has shifted to flow cytometry analysis targeting CD34+ CD38- leukemic cells due to their effectiveness in minimal/measurable residual disease detection and prognosis prediction. Nevertheless, the immunophenotype and cell frequency of these cells in the bone marrow, in the absence of leukemic cells, remains unknown. We aimed to evaluate detailed characteristics of CD34+ CD38- cells in both normal and leukemic cells by flow cytometry. METHODS: We compared the cell frequency and immunophenotype of the CD34+ CD38- fraction in the following groups: patients with idiopathic thrombocytopenic purpura and malignant lymphoma as controls (n = 17), post-treatment patients without abnormal blasts (n = 35), and patients with myeloid malignancies (n = 86). The comparison was based on the presence or absence of CD45RA expression, a marker commonly used to prospectively isolate lymphoid-primed cell populations within the CD34+ CD38- fraction. RESULTS: The CD34+ CD38- CD45RA+ cell population exhibited a significant expansion in bone marrow without leukemic cells 1 month after cord blood transplantation and in various type of myeloid malignancies, compared to the control group (p < 0.01). Continuous CD45RA expression and notable expansion of the CD34+ CD38- CD45RA- population were exclusively observed in myelodysplastic syndrome-related diseases. The CD34+ CD38- CD45RA+ population displayed frequent expression of various markers in both leukemic and non-leukemic cells, in contrast to the CD34+ CD38- CD45RA- population. CONCLUSIONS: The CD34+ CD38- fraction should be carefully evaluated considering the nature of normal hematopoietic precursor cells, their cell frequency and immunophenotype, including CD45RA expression pattern, for improving the accuracy of myeloid malignancy diagnosis.

CLINICOPATHOLOGICAL ANALYSIS OF SECONDARY RETINAL VASOPROLIFERATIVE TUMOR/REACTIVE RETINAL ASTROCYTIC TUMOR SUCCESSFULLY TREATED BY ENDORESECTION.

PURPOSE: To report the clinicopathological findings of retinal vasoproliferative tumor/reactive retinal astrocytic tumor (VPT/RRAT) with retinal vasculitis, treated by tumor resection. METHODS: A retrospective single case report. PATIENT: A 29-year-old Japanese woman was referred with cystoid macular edema and retinal vasculitis in the right eye. Best-corrected visual acuity was 0.9. Results of fundus examination, optical coherence tomography, and fluorescein angiography demonstrated VPT/RRATs in the temporal retina surrounded by a subretinal exudate, serous retinal detachment and macular edema, and retinal vasculitis. Despite 3 months of oral prednisolone treatment, a full-thickness macular hole developed. Pars plana vitrectomy and endoresection of the VPT/RRATs were performed. Pathologic and immunohistochemical analyses with anti-CD34 antibody, antiglial fibrillary acidic protein antibody, anti-Ki67 antibody, and anti-vascular endothelial growth factor antibody were performed on the excised tissue. Inflammation was evaluated by immunohistological staining with leukocyte common antigen (LCA), anti-CD3 antibody, and anti-CD20 antibody. RESULTS: After surgery, the macular hole closed, best-corrected visual acuity improved to 1.2, retinal vasculitis was ameliorated, and retinal exudate disappeared. There was no recurrence of VPT/RRAT or retinal vasculitis. Pathologic examination showed that antiglial fibrillary acidic protein and anti-vascular endothelial growth factor were widely expressed, irrespective of the distribution of blood vessels. Ki67-positive proliferating cells were detected in the perivascular area. Leukocyte common antigen-positive leukocytes and CD3-positive T cells were detected throughout the samples, whereas CD20-positive B cells were rarely detected. CONCLUSION: Endoresection of VPT/RRAT could be a good treatment option for secondary VPT/RRAT accompanied by retinal vasculitis. Pathologic findings revealed for the first time that inflammatory cells infiltrate the tissue in secondary VPT/RRAT.

The signature and predictive value of immune parameters in patients with secondary hemophagocytic lymphohistiocytosis.

BACKGROUND: Secondary hemophagocytic lymphohistiocytosis (sHLH) is a rare but fatal clinical syndrome, characterized by severe immune dysfunction and overwhelming inflammatory response. However, the host immune signature and also its role in predicting the clinical outcome are not fully described. OBJECTIVE: The present study aims to investigate the host immune status of sHLH patients in the early stage of the disease, including lymphocyte subsets, phenotypes and cytokines, and also to explore its clinical value in prognosis. METHODS: Sixty-four patients with sHLH admitted to a tertiary hospital in central China between 2018 and 2022 were enrolled, of which 21 were deceased. The subsets and phenotypes of lymphocytes, and the levels of cytokines in serum were analyzed. RESULTS: In patients with sHLH, the percentages of total T cells, CD8+ T cells, HLA-DR+ T cells, HLA-DR+CD8+ T cells, CD45RO+CD4+ T cells, and the levels of IL-1ß, IL-2R, IL-6, IL-8, IL-10 and TNF-α were significantly increased, while the percentages of CD4+ T cells, NK cells, CD45RA+CD4+ T cells, CD45RA+ regulatory T (Treg) cells, the counts of total T cells, total B cells, CD4+ T cells, CD8+ T cells, NK cells, and the ratio of CD4+ T/CD8+ T cells were significantly decreased, compared with healthy controls (HC). In addition, dysregulation of host immune response and high inflammatory status were more obvious in deceased patients than that of survivors. Kaplan-Meier survival analysis and multivariate logistic regression analysis demonstrated that lower levels of CD4+ T cells count and CD28+CD4+ T cells percentage, but higher levels of NK cells percentage and IL-1ß were poor prognostic indicators of sHLH. CONCLUSION: The evaluation of immunological markers has critical value for selecting prognostic markers and potential treatment target among adults with sHLH.

HPV-associated head and neck cancer is characterized by distinct profiles of CD8+ T cells and myeloid-derived suppressor cells.

Patients with HPV--localized head and neck cancer (HNC) show inferior outcomes after surgery and radiochemotherapy compared to HPV-associated cancers. The underlying mechanisms remain elusive, but differences in immune status and immune activity may be implicated. In this study, we analyzed immune profiles of CD8+ T cells and myeloid-derived suppressor cells (MDSC) in HPV+ versus HPV- disease.The overall frequency of CD8+ T cells was reduced in HNC versus healthy donors but substantially increased after curative therapy (surgery and/or radiochemotherapy). In HPV+ patients, this increase was associated with significant induction of peripheral blood CD8+/CD45RA-/CD62L- effector memory cells. The frequency of HPV-antigen-specific CD8+ cells was low even in patients with virally associated tumors and dropped to background levels after curative therapy. Pre-therapeutic counts of circulating monocytic MDSC, but not PMN-MDSC, were increased in patients with HPV- disease. This increase was accompanied by reduced fractions of terminally differentiated CD8+ effector cells. HPV- tumors showed reduced infiltrates of CD8+ and CD45RO+ immune cells compared with HPV+ tumors. Importantly, frequencies of tumor tissue-infiltrating PMN-MDSC were increased, while percentages of Granzyme B+ and Ki-67+ CD8 T cells were reduced in patients with HPV- disease.We report differences in frequencies and relative ratios of MDSC and effector T cells in HPV- HNC compared with more immunogenic HPV-associated disease. Our data provide new insight into the immunological profiles of these two tumor entities and may be utilized for more tailored immunotherapeutic approaches in the future.

PD-1- CD45RA+ effector-memory CD8 T cells and CXCL10+ macrophages are associated with response to atezolizumab plus bevacizumab in advanced hepatocellular carcinoma.

The combination of atezolizumab plus bevacizumab (atezo/bev) has dramatically changed the treatment landscape of advanced HCC (aHCC), achieving durable responses in some patients. Using single-cell transcriptomics, we characterize the intra-tumoural and peripheral immune context of patients with aHCC treated with atezo/bev. Tumours from patients with durable responses are enriched for PDL1+ CXCL10+ macrophages and, based on cell-cell interaction analysis, express high levels of CXCL9/10/11 and are predicted to attract peripheral CXCR3+ CD8+ effector-memory T cells (CD8 TEM) into the tumour. Based on T cell receptor sharing and pseudotime trajectory analysis, we propose that CD8 TEM preferentially differentiate into clonally-expanded PD1- CD45RA+ effector-memory CD8+ T cells (CD8 TEMRA) with pronounced cytotoxicity. In contrast, in non-responders, CD8 TEM remain frozen in their effector-memory state. Finally, in responders, CD8 TEMRA display a high degree of T cell receptor sharing with blood, consistent with their patrolling activity. These findings may help understand the possible mechanisms underlying response to atezo/bev in aHCC.

PTPRC functions as a prognosis biomarker in the tumor microenvironment of cutaneous melanoma.

Cutaneous melanoma is one of the most malignant types of skin cancer, with an extremely poor prognosis. Immune cells infiltrated in the tumor microenvironment (TME) affects melanoma initiation, progression, prognosis and immunotherapy strategies in melanoma. The potential utility of TME-related genes as a prognostic model for melanoma and as a predictor of immunotherapeutic response merits further exploration. In this study, we determined that an immune-related gene, protein tyrosine phosphatase receptor type C (PTPRC), was positively correlated with the positive prognosis of melanoma patients. Integration of this gene with TNM classification created a predictive model that showed better performance in determining overall survival than others. PTPRC expression was positively correlated with the levels of immune checkpoint molecules, and PTPRC knockdown significantly enhanced the migration, invasion, and proliferation of melanoma cells. Finally, immunohistochemical results from HPA and Real-time quantitative PCR of clinical tissues confirmed that PTPRC expression was higher in melanoma than in normal skin. In conclusion, PTPRC served as a potential predictor of survival and response to immunotherapy in melanoma patients. The risk model combining the PTPRC and TNM classifications holds the potential to be a promising tool for prognostic prediction of cutaneous melanoma. This will help in the effective clinical management of melanoma patients.