Loss Of CD4 Research Articles

HIV associated cell death: Peptide-induced apoptosis restricts viral transmission.

The human immunodeficiency virus (HIV) is still a global pandemic and despite the successful use of anti-retroviral therapy, a well-established cure remains to be identified. Viral modulation of cell death has a significant role in HIV pathogenesis. Here we sought to understand the major mechanisms of HIV-induced death of lymphocytes and the effects on viral transmission. Flow cytometry analysis of lymphocytes from five latent HIV-infected patients, and HIV IIIB-infected MT2 cells demonstrated both necrosis and apoptosis to be the major mechanisms of cell death in CD4+ and CD4-/CD8- lymphocytes. Significantly, pro-apoptotic tumor necrosis factor (TNF) peptide (P13) was found to inhibit HIV-related cell death and reduced viral transmission. Whereas pro-necrotic TNF peptide (P16) had little effect on HIV-related cell death and viral transmission. Understanding mechanisms by which cell death can be manipulated may provide additional drug targets to reduce the loss of CD4+ cells and the formation of a viral reservoir in HIV infection.

Dynamic changes in thymic sub-populations during acute and long-term infections with virulent and virulence-attenuated Salmonella Typhimurium strains in C57BL/6 and autoimmune-prone lpr mice

SalmonellaTyphimurium infection in mice results in drastic loss of immature CD4− CD8− double negative (DN) and CD4+ CD8+ double positive (DP) thymic subsets compared to mature single positive (SP) subsets. We investigated changes in thymocyte sub-populations post infection with a wild type (WT) virulent strain and ΔrpoS, a virulence-attenuated strain, of Salmonella Typhimurium in C57BL/6 (B6) and Fas-deficient autoimmune-prone lpr mice. The WT strain caused acute thymic atrophy with greater loss of thymocytes in lpr mice compared to B6 mice. Infection with ΔrpoS caused progressive thymic atrophy in B6 and lpr mice. Analysis of thymocyte subsets revealed that immature thymocytes including the DN, immature single positive (ISP), and DP thymocytes underwent extensive loss. SP thymocytes were more resistant to loss in WT-infected B6 mice, whereas WT-infected lpr and ΔrpoS-infected mice exhibited depletion of SP thymocytes. Overall, thymocyte sub-populations exhibited differential susceptibilities depending on bacterial virulence and the host background.

Response to Chemoimmunotherapy Is Associated With Expansion of Systemic Antitumor CD4 + Th1 Response in Metastatic Non-Small Cell Lung Cancer.

Limited data have reported the evolution of antitumor immune responses under chemoimmunotherapy (chemo-IO) in patients with metastatic non-small cell lung cancer. In this concise study, we performed dynamic monitoring of antitumor CD4 + T helper 1 (Th1) response in peripheral blood from 12 patients receiving a first-line chemo-IO. Tumor-reactive CD4 + Th1 cells were assessed within blood lymphocytes using interferon-gamma enzyme-linked immunospot assay to detect telomerase (TERT)-specific T cells at baseline, 3 and 12 months after treatment. An induction of circulating anti-TERT CD4 + Th1 response were found in 6 of 12 patients at 3 months after chemo-IO. In contrast, 3 patients had a substantial decrease in their preexisting response and 3 remained nonimmune responders. Among patients with chemo-IO-induced immune response, half achieved an objective clinical response and had long-lasting circulating anti-TERT CD4 + Th1 cells detected for at least 1 year. In contrast, no objective response was documented in nonimmune responders and a link between the loss of anti-TERT CD4 + Th1 responses were observed in patients with progressive disease. This preliminary work supports a relationship between the efficacy of combinatorial chemo-IO and circulating anti-TERT CD4 + Th1 responses and highlights the interest to implement blood-based monitoring of tumor-reactive CD4 + T cells that could be additional help for patient management.

Abstract TMP32: Genetic Knockout Of CD38 Is Associated With Improved Cognitive Performance And Protection Against Oxidative Stress In Aged Mice

Introduction: CD38 enzymatic activity is the main determinant of the age-dependent decline in nicotinamide adenine dinucleotide (NAD + ) levels and the loss of CD38 function has been associated with increased longevity in rodents. Therefore, we hypothesize that the loss of CD38 and its enzymatic function will improve cognitive performance in advanced age through the preservation of NAD + levels and the protection against oxidative stress. Methods: CD38 Knockout (CD38KO) and C57BL/6J (wild type WT) male mice were aged for at least 24 months. The cognitive performance was compared through Barnes maze, Fear conditioning and Y-maze tests. Dihydroethidium (DHE), Diaminofluorescein-2 diacetate (DAF) and nicotinamide adenine dinucleotide phosphate NAD(P)H staining were used to assess the levels of superoxide, nitric oxide (NO) and NAD(P)H in the brain, respectively. Results: 5 WT and 5 CD38KO mice aged (24-30) months were included. While there were no significant differences in fear conditioning and Y-maze tests, CD38KO mice showed better memory performance in Barnes maze test including shorter distance travelled (CD38KO: 2±0.06 vs WT: 3.7±1.1 m, p=0.008), longer time in proximity of the holes (CD38KO: 51.2±11.1 vs WT: 34.6±8.4 s, p=0.027), and shorter distance from the hole (CD38KO: 0.17±0.025 vs WT: 0.23±0.04 m, p=0.038) and less errors (CD38KO: 3.5±2.3 vs WKY: 6±0.7, p=0.04). Brain tissue analysis showed 58% lower superoxide (p<0.0001), 129% higher NO (p=0.009), and 141% higher NAD(P)H levels (p<0.0001) in CD38KO compared to WT mice. Conclusions: The loss of CD38 and its enzymatic function is associated with better memory performance, less oxidative stress, and higher NAD(P)H in older mice suggesting its potential role as a future therapeutic target against age-related dementias.

A Simian Immunodeficiency Virus-Infected Macaque Model of Enterocytozoon bieneusi Infection.

Microsporidiosis caused by Enterocytozoon bieneusi is a common opportunistic infection in patients with HIV/AIDS and those on immunosuppressive therapy. A significant loss of mucosal or peripheral CD4+ T cells and subsequent dysfunction of the intestinal immune system may be responsible for the development of chronic microsporidiosis in these patients. We have used the Simian immunodeficiency virus (SIV)-infected macaque model to investigate this relationship. To establish the course of E. bieneusi infection in SIV-infected animals, four SIV-infected animals were experimentally challenged with E. bieneusi when their CD4+ counts dropped to less than 300 cells/μL of blood. Analysis of fecal samples by nested polymerase chain reaction revealed that three out of four E. bieneusi-infected macaques continued to shed spores for 7-24 months after infection, an indication of chronic microsporidiosis. Four other SIV-infected macaques, after having an initial negative phase, spontaneously acquired E. bieneusi infection when their CD4+ counts dropped to less than 600 cells/μL of blood and shed spores for 8-19 months. The shedding of E. bieneusi spores in the feces increased relative to decrease in peripheral blood CD4+ T cell numbers. Gut biopsies were obtained before and after challenge to phenotype the mucosal lymphocyte subsets using flow cytometry. The immunophenotypic analysis showed no restoration of CD4+ T cells after E. bieneusi infection in the intestinal cells. A slight increase in the percentage population of CD4+ T cells in peripheral blood did not have any effect on the control of E. bieneusi infection in the SIV-infected macaques. These preliminary studies demonstrate that SIV-infected macaques develop chronic E. bieneusi infections as their CD4+ counts dropped to below 300 cells/μL of blood.

A stem cell epigenome is associated with primary nonresponse to CD19 CAR T cells in pediatric acute lymphoblastic leukemia

AbstractCD19 chimeric antigen receptor T-cell therapy (CD19-CAR) has changed the treatment landscape and outcomes for patients with pre–B-cell acute lymphoblastic leukemia (B-ALL). Unfortunately, primary nonresponse (PNR), sustained CD19+ disease, and concurrent expansion of CD19-CAR occur in 20% of the patients and is associated with adverse outcomes. Although some failures may be attributable to CD19 loss, mechanisms of CD19-independent, leukemia-intrinsic resistance to CD19-CAR remain poorly understood. We hypothesize that PNR leukemias are distinct compared with primary sensitive (PS) leukemias and that these differences are present before treatment. We used a multiomic approach to investigate this in 14 patients (7 with PNR and 7 with PS) enrolled in the PLAT-02 trial at Seattle Children’s Hospital. Long-read PacBio sequencing helped identify 1 PNR in which 47% of CD19 transcripts had exon 2 skipping, but other samples lacked CD19 transcript abnormalities. Epigenetic profiling discovered DNA hypermethylation at genes targeted by polycomb repressive complex 2 (PRC2) in embryonic stem cells. Similarly, assays of transposase-accessible chromatin–sequencing revealed reduced accessibility at these PRC2 target genes, with a gain in accessibility of regions characteristic of hematopoietic stem cells and multilineage progenitors in PNR. Single-cell RNA sequencing and cytometry by time of flight analyses identified leukemic subpopulations expressing multilineage markers and decreased antigen presentation in PNR. We thus describe the association of a stem cell epigenome with primary resistance to CD19-CAR therapy. Future trials incorporating these biomarkers, with the addition of multispecific CAR T cells targeting against leukemic stem cell or myeloid antigens, and/or combined epigenetic therapy to disrupt this distinct stem cell epigenome may improve outcomes of patients with B-ALL.

Leishmania donovani induces CD300a expression to dampen effector properties of CD11c+ dendritic and antigen activated CD8+ T cells

CD8+ T cells are an important regiment of adaptive immunity that play a decisive role in elimination of many species of Leishmania parasite from the host. In visceral leishmaniasis, caused by L. donovani, the loss of CD8+ T cells function has been found associated with augmented pathogenesis. The factors determining CD8+ T cells activation and function against Leishmania antigens are largely unknown. In this study, we investigated the role of an immune inhibitory receptor, CD300a, on the effector properties of dendritic cells and CD8+ T cells. We observed that the Leishmania regulates the effectors function of CD8+ T cells by increasing CD300a expression on CD11c+ dendritic cells. The abrogation of CD300a signaling in parasites infected animals induced CD8+ T cell abilities to produce IFN-γ, TNF-α and also helped them to acquire desired multifunctionality. The CD300a receptor blocking also enhanced the number of CD8+ T cells memory phenotypes at the early days of infection, suggesting its potential beneficial role in vaccine induced immunity. We also observed significantly enhanced levels of pro-inflammatory cytokines in the spleen of CD300a blocked infected animals with concomitant reduced spleen parasite load. Additionally, the abrogation of CD300a signals in the infected animals helped in establishing Th1 type protective humoral immunity with significantly elevated levels of IgG2a antibodies. Since CD8+ T cells are an important determinant of vaccine induced immunity against leishmaniasis, the findings corroborate the potential of CD300a in vaccine induced immunity and thus require further attention.

Molecular Imaging of Oxygenation Changes during Immunotherapy in Combination with Paclitaxel in Triple Negative Breast Cancer.

Hypoxia is a common feature of the tumor microenvironment, including that of triple-negative breast cancer (TNBC), an aggressive breast cancer subtype with a high five-year mortality rate. Using [18F]-fluoromisonidazole (FMISO) positron emission tomography (PET) imaging, we aimed to monitor changes in response to immunotherapy (IMT) with chemotherapy in TNBC. TNBC-tumor-bearing mice received paclitaxel (PTX) ± immune checkpoint inhibitors anti-programmed death 1 and anti-cytotoxic T-lymphocyte 4. FMISO-PET imaging was performed on treatment days 0, 6, and 12. Max and mean standard uptake values (SUVmax and SUVmean, respectively), histological analyses, and flow cytometry results were compared. FMISO-PET imaging revealed differences in tumor biology between treatment groups prior to tumor volume changes. 4T1 responders showed SUVmean 1.6-fold lower (p = 0.02) and 1.8-fold lower (p = 0.02) than non-responders on days 6 and 12, respectively. E0771 responders showed SUVmean 3.6-fold lower (p = 0.001) and 2.7-fold lower (p = 0.03) than non-responders on days 6 and 12, respectively. Immunohistochemical analyses revealed IMT plus PTX decreased hypoxia and proliferation and increased vascularity compared to control. Combination IMT/PTX recovered the loss of CD4+ T-cells observed with single-agent therapies. PET imaging can provide timely, longitudinal data on the TNBC tumor microenvironment, specifically intratumoral hypoxia, predicting therapeutic response to IMT plus chemotherapy.

Inhibition of DDX3X ameliorated CD4+ T cells pyroptosis and improves survival in septic mice

Over-expression of DDX3X mRNA is associated with T cell loss in septic patients. This study aimed to investigate the molecular mechanism of DDX3X on T cell reduction in sepsis. The sepsis model was established using lipopolysaccharide stimulation in vitro and cecal ligation and puncture (CLP) surgery in vivo. Results showed that the expression of DDX3X was significantly upregulated in CD4+ T cells in sepsis. RK-33, the inhibitor of DDX3X, was found to dramatically increase CD4+ T cell counts and prolong the survival rate of mice with sepsis. The results also showed that the expression of caspase-1/GSDMD in CD4+ T cells was significantly increased in vitro and in vivo, and RK-33 can substantially reduce CD4+ T cell pyroptosis through inhibiting NLRP3/caspase-1/GSDMD. Globally, our results suggest that DDX3X is involved in the loss of CD4+ T cells partly through activating the pyroptotic pathway during sepsis, which may provide potential targets for therapeutic interventions in this highly lethal disease.

20P Human virome epitope-level antiviral antibody profiling identified the cytomegalovirus (CMV) as the main driver of senescent immune phenotype (SIP) in patients with advanced non-small cell lung cancer (aNSCLC)

Immunosenescence is a progressive remodeling of immune functions with a multifactorial etiology including aging and chronic antigenic stressors (inflammation, infections, cancer). We showed that a high pretreatment SIP (CD28–CD57+KLRG1+CD8+ circulating T cells>39.5%, SIP+) was associated with resistance to ICB in patients with aNSCLC. Chronic viral infections may speed up immune aging, especially CMV which affects blood T cells phenotype (loss of CD28, overexpression of CD57). We aimed to assess antiviral serological profile and its association with SIP status.

Impact of CD4 T cells on intratumoral CD8 T-cell exhaustion and responsiveness to PD-1 blockade therapy in mouse brain tumors

BackgroundGlioblastoma is a fatal disease despite aggressive multimodal therapy. PD-1 blockade, a therapy that reinvigorates hypofunctional exhausted CD8 T cells (Tex) in many malignancies, has not shown efficacy in glioblastoma....

Autophagy inducer rapamycin treatment reduces IFN-I-mediated Inflammation and improves anti-HIV-1 T cell response in vivo.

A hallmark of HIV-1 infection is chronic inflammation, even in patients treated with antiretroviral therapy (ART). Chronic inflammation drives HIV-1 pathogenesis, leading to loss of CD4+ T cells and exhaustion of antiviral immunity. Therefore, strategies to safely reduce systematic inflammation are needed to halt disease progression and restore defective immune responses. Autophagy is a cellular mechanism for disposal of damaged organelles and elimination of intracellular pathogens. Autophagy is pivotal for energy homeostasis and plays critical roles in regulating immunity. However, how it regulates inflammation and antiviral T cell responses during HIV infection is unclear. Here, we demonstrate that autophagy is directly linked to IFN-I signaling, which is a key driver of immune activation and T cell exhaustion during chronic HIV infection. Impairment of autophagy leads to spontaneous IFN-I signaling, and autophagy induction reduces IFN-I signaling in monocytic cells. Importantly, in HIV-1-infected humanized mice, autophagy inducer rapamycin treatment significantly reduced persistent IFN-I-mediated inflammation and improved antiviral T cell responses. Cotreatment of rapamycin with ART led to significantly reduced viral rebound after ART withdrawal. Taken together, our data suggest that therapeutically targeting autophagy is a promising approach to treat persistent inflammation and improve immune control of HIV replication.

A Multi-Center Study: Outcomes Among Relapsed/Refractory Diffuse Large B Cell Lymphoma Patients with CD20 Loss

Background: Despite novel therapies for relapsed/refractory (R/R) DLBCL, outcomes among this cohort remain poor. A prior study suggested that CD20 loss is a mechanism of therapeutic resistance and disease progression in patients treated with rituximab. Whether CD20 loss leads to inferior outcomes is unknown. We hypothesized that CD20 loss in the setting of R/R DLBCL is associated with inferior survival and response rates. Methods: We performed a retrospective cohort study of R/R DLBCL patients seen at the University of Michigan, Mayo Clinic and University of Illinois Hospital between 2003-2021. Inclusion criteria were patients ≥ 18 years old, diagnosis of CD20 positive (+) DLBCL (de novo or transformed), relapsed/refractory disease, and CD20 status available at relapse on flow cytometry or immunohistochemistry. CD20 status was reviewed by the pathology department of each individual institution without central review. Overall survival (OS) was defined as time from first relapse to death and event-free survival (EFS) was time from first relapse to death or next treatment; both of which were estimated with Kaplan Meier analysis. Overall response rate (ORR) was complete response or partial response after first salvage therapy. Descriptive statistics with univariate and multivariate analyses were also performed. Results: 129 patients were included (64% male) with median age at diagnosis of 62 years. Majority of patients had stage III/IV disease at diagnosis (85%) and 15% had transformed DLBCL. At diagnosis, all patients had CD20+ disease. Median time from diagnosis to first relapse was 21.9 months (interquartile range 9.6, 53.6). Among the 129 patients, 96 were classified as CD20 positive at their time of first relapse (84 from Michigan, 12 from UIC) and 33 were classified as CD20 negative (12 from Michigan, 4 from UIC, and 17 from Mayo). The estimated median time from first relapse to death in the CD20+ group was 17.9 months (95% CI 14.0, 39.7) compared to 12.0 months (95% CI 9.9, 42.2) among the CD20 negative group. An overall (unstratified) log-rank test suggested there were no differences in OS between CD20 groups (p = 0.311).The estimated median EFS in the CD20+ group was 3.8 months (95% CI 3.3, 4.3) compared to 3.0 months (95% CI 2.5, 4.3) in the CD20 negative group, this difference was not statistically significant (p = 0.063). Of patients who became CD20 negative at relapse, 79% received anti-CD20 directed therapy thereafter, as part of salvage or with conditioning prior to transplant. Conclusion: Patients with DLBCL who relapse and maintain CD20 positivity seem to have a trend towards improved EFS and OS, though this was not statistically significant. We also found that for the majority of patients, loss of CD20 did not alter use of anti-CD20 directed therapy. Our study was limited given the retrospective data collection, variation in data collection methodology among institutions and small sample size which may have limited our ability to detect a statistical significance in outcomes. We will plan on expanding our data set in a future analysis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Overcoming CD19-Negative Relapses in Patients with B-Cell Lymphomas Treated with Tisagenlecleucel

Anti-CD19 chimeric antigen receptor T cell (CART) immunotherapy has revolutionized the treatment of large B-cell lymphomas (LBCL). However, up to 70% of LBCL patients (pts) ultimately fail CART treatment. Several mechanisms of relapse have been described, such as T cell exhaustion, an immunosuppressive tumor microenvironment, and antigen-negative escape. While antigen-negative relapse after anti-CD19 CART (CART19) is well studied in acute B lymphoblastic leukemia, less data are available for LBCL, especially in pts treated with the 4-1BB costimulated CART19, tisagenlecleucel (tisa-cel, formerly CTL019). In this study, we evaluated the frequency of CD19 loss in LBCL pts treated with tisa-cel and identified CD79 as an antigen highly expressed by immunohistochemistry (IHC) in CD19-negative biopsies. We then generated and optimized an anti-CD79 CART (CART79) and subsequently cloned a dual anti-CD79b/anti-CD19 CART that showed high potency against CD19-neg models. We first studied an exploratory cohort of 38 pts treated at the University of Pennsylvania on the first single-center clinical trial of CTL019 for B-cell lymphomas (NCT02030834 and Chong, NEJM, 2021). Among relapsed or refractory pts (n=23, 60.5%), 17 pts had a biopsy at disease progression (PD). We analyzed CD19 and CD79a expression in these biopsies by IHC. Four biopsies (23.5%) showed no CD19 expression by neoplastic cells. Also, 12 pts had available matched biopsies pre-CTL019 infusion (performed within 1 year before infusion) and at PD. We observed that 3/12 (25%) pts converted from a CD19-positive to a CD19-negative tumor after CTL019. Notably, 100% of biopsies at PD showed high CD79a expression. We then studied a confirmatory cohort including 110 LCBL pts treated with commercial tisa-cel. In this retrospective cohort, 63 pts had PD, and 31 had an available biopsy at PD. In line with previous observations, 10/26 (38%) showed loss of CD19 at PD, and, of note, 5/23 (22%) loss of CD20, a B-cell marker targeted by previous therapies. Again, 100% of pts maintained CD79a expression. (Figure A). These data confirm that loss of detection of CD19 at the level of sensitivity of IHC is frequently observed at PD and may be a cause of resistance in LBCL pts treated with tisa-cel. In addition, persistence of CD79 expression suggests that it may be an optimal therapeutic target for potentially preventing CD19-negative PD. Therefore, we aimed to generate and optimize a CART79 product. We cloned and screened a large panel of anti-CD79 CAR constructs (n=32) generated using 6 different scFvs (targeting either CD79a or CD79b), two scFv chain orientations (H to L vs. L to H), three hinges (derived from CD8a, IgG4, or truncated IgG4). We used the CD8 transmembrane and 4-1BB co-stimulatory domains of tisa-cel, given its established clinical safety and efficacy profile. The constructs were initially screened in-vitro against LBCL cells (OCI-Ly18), evaluating cytotoxicity, proliferation, and cytokine production. The six top candidates were then tested in-vivo using human xenograft models (NSG mice subcutaneously engrafted with OCI-Ly18 cells). When the tumor was palpable, mice were randomized to receive one dose (2.5x106/mouse) of CART79 candidates, CART19, or untransduced T cells (UTD) intravenously. An anti-CD79b CAR construct (CART79#3: H-L orientation, truncated IgG4 hinge), demonstrated the strongest anti-tumor effect. This construct was incorporated into a bicistronic vector also containing anti-CD19 CAR (FMC63-based). We tested the activity of this novel anti-CD19/CD79b dual CART in-vitro, using CRISPR/Cas9 gene editing to knock out (KO) either CD19 or CD79b in HBL-1 LBCL cells. CD19 KO and CD79b KO HBL-1 cells were mixed 1:1 to simulate the heterogeneity of lymphomas and tumor targeting was tested for CART19, CART79b, dual CART79b/19, and UTD. In both in-vitro and in-vivo models, dual CART19/79b demonstrated a strong anti-tumor effect, possibly preventing or treating CD19-negative escape (Figure B). In conclusion, our study demonstrates that CD19 loss by IHC is a marker of treatment failure in tisa cel-treated LBCL pts. We also demonstrate that CD79, as opposed to CD20, is expressed in virtually all cases of PD and is an ideal target for next-generation CART immunotherapy. Finally, we developed a dual CART19/79b that shows high activity against CD19-negative disease progression. This construct will be taken to phase I clinical studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Rapid Tumor Debulking of Relapsed/Refractory CLL Patients By PI3Kδ Inhibition and Anti-CD20 Monoclonal Antibody Treatment

Introduction Effective multi-targeted drug combinations could rapidly achieve deep remissions in CLL allowing for defined duration treatment. The combination of a novel highly-specific phosphoinositide 3-kinase delta (PI3Kδ) inhibitor and casein kinase 1 epsilon (CSK1ε) inhibitor, umbralisib (UMB), and a glycoengineered chimeric monoclonal antibody (mAb) targeting a unique epitope on CD20, ublituximab (UBL), with the BCL2 inhibitor venetoclax (VEN) may decrease drug resistance, reduce risk of tumor lysis syndrome (TLS) and achieve higher levels of undetectable minimal residual disease (MRD). This triple agent therapy is being evaluated in relapsed or refractory CLL patients (U2-VEN, ClinicalTrials.gov NCT03379051). Patients received oral 800 mg UMB once-daily. For Cycle 1, UBL was given via IV on Day 1 (150 mg), Day 2 (750 mg), Day 8 (900 mg), Day 15 (900 mg), and then on Day 1 in Cycles 2 and 3 (900 mg); for 3 cycles of 28 days, followed by consolidation with UMB and VEN for cycles 4 to 24. Patient (n = 25) blood samples were collected Pre- and Post-treatment on days of UBL administration during cycles 1-3. Our studies focused on UMB and UBL treatment effects on: 1) The decrease in the circulating CLL tumor burden and thus the subsequent risk of TLS from the addition of VEN to the regimen; and 2) CD20 antigen loss on CLL cells, a major mechanism of anti-CD20 mAb resistance. Methods Blood was analyzed by clinical laboratory automated cell counter and then stained with antibodies against CD3, CD5, CD19, and CD20 for flow cytometry analysis. CLL cell counts were calculated from flow cytometry and clinical laboratory results. To quantitate CD20 surface molecules, peripheral blood mononuclear cells were isolated and stained for flow cytometry analysis with the addition of a Molecules of Equivalent Soluble Fluorochrome standard curve. All human specimen collection and usage was conducted with written informed consent after approval of our Institutional Research Subjects Review Board according to the ethical guidelines of the Declaration of Helsinki. Results Average CLL cell counts (47 x 109/L) decreased rapidly after administration of the first dose of UMB and UBL (C1D1 Post, 26 x 109/L), which rebounded the next day (C1D2 Pre, 43 x 109/L) and then decreased after the second treatment (C1D2 Post, 24 x 109/L). By Day 15, the average CLL cell count had dropped to 9 x 109/L, which decreased further to 5 x 109/L by the start of cycle 3 (Fig. 1). Notably, the variation in CLL cell counts (error bars) decreases over treatment time, suggesting that most CLL patients' tumor burdens are decreasing to a similar low level independent of starting CLL cell count. The change in average CLL cell count between Pre and Post measurements is initially very high (45% decrease C1D1) with decreasing effectiveness over treatment time (8% decrease C3D1). This change could be due to the loss of CLL cell membrane CD20 levels leading to mAb resistance. Pre-treatment (C1D1 Pre) average CLL cell membrane CD20 levels (n=18) were 29,000 CD20 molecules/cell. This decreased to 9,000 molecules/cell after the first treatment (C1D1 Post), stabilized at 9,100 molecules/cell on the next day (C1D2 Pre) and then decreased further after the second treatment (C1D2 Post) to 1600 molecules/cell. After day 8 treatment, CD20 levels remained at 1,000 molecules/cell or less until the end of this study (C3D1). Discussion UMB and UBL rapidly decrease circulating CLL counts over three cycles of treatment, reducing most CLL patient tumor burden to similar low levels. This clinical response could facilitate addition of VEN in the 4thcycle of therapy by decreasing the risk of TLS. The rapid loss of surface CD20 from CLL cells to 1,000 CD20 molecules/cell or less by C1D8 Post has the potential to cause acquired resistance to anti-CD20 mAb therapy. This rapid and sustained loss of CD20 levels is consistent with that reported at standard doses for other anti-CD20 mAbs. However, an effect of concomitant UMB on CD20 levels cannot be ruled out. Previous work suggests that lower anti-CD20 mAb doses can prevent these large decreases in CD20 levels and increase UBL efficacy. In conclusion, our initial data show that treatment with a selective PI3Kδ and CSK1ε inhibitor (UMB) combined with an anti-CD20 mAb (UBL) is effective in decreasing CLL cell counts in all patients assessed in the U2-VEN clinical trial. However, treatment efficacy could be limited by large decreases in the CLL surface CD20 levels. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Resistance to CD19 Immunotherapy in B-Cell ALL Is Associated with Loss of CD19 and CD22 and an Enhanced Vulnerability to B Cell Receptor Signaling Inhibition

Resistance to CD19 Immunotherapy in B-Cell ALL Is Associated with Loss of CD19 and CD22 and an Enhanced Vulnerability to B Cell Receptor Signaling Inhibition

Analysis of Immune Pharmacodynamic and Baseline Biomarkers in Patients with Relapsed or Refractory Follicular Lymphoma Treated with Mosunetuzumab in Combination with Lenalidomide

Background: Mosunetuzumab (M) is a bispecific monoclonal antibody targeting CD20 and CD3 that redirects T cells to eliminate B cells and has shown efficacy in patients (pts) with relapsed/refractory (R/R) follicular lymphoma (FL; Budde et al. Lancet Oncol 2022). Lenalidomide (Len) is clinically active in FL, and its immunomodulatory activity offers potential synergistic efficacy when combined with M. Here, we present preliminary data from a Phase Ib/II trial (NCT04246086), which aimed to characterize pharmacodynamic biomarkers and investigate baseline (BL) features of early progressive disease (PD), in pts with R/R FL treated with M+Len. Methods: Eligible pts had Grade 1-3a R/R FL and received ≥1 prior systemic anti-cancer therapy. Pts received ≤12 cycles of M+Len (Cycle [C] 1: 21 days; C2-12: 28 days). In C1, step-up doses of intravenous M were given on C1 Day (D) 1 (1mg) and C1D8 (2mg), with the target dose (30mg) given on C1D15 and then on D1 of C2‒12. Len (20mg) was administered orally on D2‒22 (n=10) and on D1-21 of C2-12 for the remaining 19 pts. Activation of immune cells during M+Len treatment was assessed in longitudinal collections of peripheral blood by flow cytometry, and plasma IL-6 levels were evaluated by enzyme-linked immunosorbent assay. CD20 expression was assessed by immunohistochemistry in BL and PD biopsies. CD20 loss was defined as ≤5% CD20+PAX5+ cells. Mutation profiling was performed by next-generation sequencing (NGS). A modified version of the AVENIO circulating tumor (ct)DNA analysis workflow and pipeline (Roche; for research use only) was used for NGS, based on the previously described CAPP-Seq technology (Kurtz et al. J Clin Oncol 2018). Results: A transient reduction in circulating T-cell counts (likely due to margination) and increase in percentage (%) of T cells expressing the activation marker CD69 were observed after M monotherapy in C1, and these kinetics were maintained in subsequent cycles, particularly in the CD4 T-cell population. Maturation of T cells, measured as % of T cells expressing human leukocyte antigen DR, increased after C2, following the addition of Len, and was sustained throughout M+Len treatment. The % of circulating programmed cell death protein 1 (PD-1)+ CD4 T cells increased after the first cycle of M, implying a rapid activation of T cells by M, and progressively decreased after C2. A decrease in % of PD1+ CD4 T cells suggested that sustained PD-1 expression, often a sign of T cell exhaustion, does not occur during M+Len treatment. Natural killer (NK) cells showed transient activation following M infusion (assessed as margination and % of CD69+ NK cells), and a sustained increase in functional markers, like granzyme B, during treatment with M+Len. IL-6 secretion was often enhanced after the first M infusion in C1. However, increased IL-6 levels were not observed after M+Len co-administration in subsequent cycles. As of April 25, 2022, 29 pts had been enrolled and were evaluable for efficacy. Best objective response rate was 89.7%, with complete metabolic response (CMR) observed in 21 pts (72.4%). Five cases of early PD (≤6 cycles of treatment) were observed. BL CD20 expression was heterogeneous (n=25 BL biopsies), and one CD20-negative pt experienced early PD. Two pts with early PD showed CD20 loss at progression. ctDNA analysis was performed in a subgroup of 5 pts with PD vs 5 with CMR for signal seeking. A trend towards higher median ctDNA levels at BL, measured as mutant molecules per mL, was observed in pts with early PD (400.1) vs CMR (58.6). Moreover, a higher median number of somatic variants was found in plasma (145.0) vs tissue (120.0) at BL, with a lower median concordance of plasma variants in tissue in pts with early PD (n=4; 43.8%) compared with those achieving CMR (n=5; 94.1%), suggesting higher tumor heterogeneity in early PD. Conclusions: In pts with R/R FL, our preliminary data suggest that M+Len activates the immune system. Increased % of T and NK cells expressing maturation and functional markers were observed during M+Len treatment. In contrast, addition of Len to M did not enhance IL-6 secretion, a cytokine known to be related with cytokine release syndrome, which is consistent with the manageable safety profile previously reported for this combination. Future investigations with a larger sample size are needed to further assess the prognostic value of CD20 expression in response to the M+Len combination and the prognostic value of ctDNA measurement at BL.

Role of TNFRSF17 and GPRC5D Structural and Point Mutations in Resistance to Targeted Immunotherapies in Multiple Myeloma (MM)

Adaptive T cell therapy using chimeric antigen receptor (CAR) T cells and T cell engagers (TCE) targeting BCMA and GPRC5D have demonstrated very encouraging responses in heavily pre-treated multiple myeloma (MM). Recent studies interrogating T cells repertoires and fitness correlated resistance to these immune based therapies with exhausted T cell states. While in lymphoma, antigen escape with CD19 loss is observed in up to 30% of patients with acquired resistance to adaptive therapies, in MM TNFRSF17 or GPRC5D structural mutations are thought to be less prevalent in recipients of BCMA or GPRC5D-targeting CARs or TCEs . Bi-allelic loss (n=2) and a missense truncating mutation (n=1) of TNFRSF17 with CAR T cells, as well as one case of biallelic loss in TNFRSF17 with TCE are reported to date with no reported cases of bi-allelic loss of GPRC5D. Further studies of the role TNFRSF17 or GPRC5D structural or point mutations to the immune escape to these adaptive therapies are needed. Serial BM aspirates were collected from patients treated with BCMA or GPRC5D-targeting TCE or CARs prior to therapy and at relapse. MM cells were profiled with single cell mRNA (scRNAseq), single cell copy number (scCNVseq) using the 10x Genomics GemCode system and whole genome sequencing (WGS). Sequenced reads were aligned to hg38. Single cell reads were processed with CellRanger and downstream analyses with Seurat R package. In order to define the prevalence of TNFRSF17 or GRPC5D structural variants and mutations in MM, we performed WGS (n=19, 14 pre-CAR/TCE, 5 post-CAR/TCE) as well as scCNVseq (n=33, 16 RRMM, 7 pre-CAR/TCE, 8 post-CAR/TCE) on sorted CD138+ cells from relapsed MM patients. WGS (100x coverage) identified at baseline a subclonal deletion at the TNFRSF17 gene locus in only 1 patient; this small subclone subsequently drove the progression post BCMA CAR T cell therapy with an acquired biallelic loss of TNFRSF17. Mono-allelic large and focal deletions of GPRC5D (chr.12p) were detected by WGS in 7 cases (5 deletions, 2 copy neutral LOH). scCNVseq studies identified more frequent subclonal monoallelic copy number losses of TNFRSF17 in 11.2% of the cells (27884 MM cells) prior to anti-BCMA therapy exposure with the deletions ranging from 200 kb to 2.3 Mb. A homozygous deletion of TNFRSF17 was noted in 2 patients at the time of disease progression, one post CAR T and the other post BCMAxCD3 TCE. Structural mutations at the GPRC5D locus were also identified by scCNVseq with monoallelic and biallelic deletions in 11.9% and 2.7% of the cells, respectively. Clonal biallelic loss of GPRC5D post GPRC5DxCD3 TCE was detected in one patient who harbored a monoallelic loss in 79.2% of the cells pre-TCE. With regard to point mutations, in one patient progressing from a durable CR on BCMA TCE, we observed an acquired mono-allelic loss of TNFRSF17 (scCNV) coupled with de novo mutation in TNFRSF17 extracellular domain c.81G>C [p.(R27P)]. This point R27P mutation is predicted to be damaging to BCMA (Polyphen) with disruption of several H-bonds between R27 and C24, Q25, S29, S30 and T32 of BCMA extracellular aa residues. Importantly, molecular dynamic simulations of WT vs. mutant R27P using publicly deposited crystal structural of BCMA bound to anti-BCMA Ab (J22.9-xi) (PBD ID 4ZFO) demonstrated that this mutation disrupts all H-bonds between BCMA and the light chain of chimeric mouse/human anti-BCMA Ab (J22.9-xi). Functionally, primary CD138+ cells with mutant BCMA [p.(R27P)] as well as K562 cells transduced with mutant BCMA [p.(R27P)] were completely resistant to BCMAxCD3 cytotoxicity with teclistamab and elranatamab compared to cells with WT BCMA. While structural alterations of TNFRSF17 and GPRC5D remain relatively rare, we have identified biallelic loss of TNFRSF17 post BCMA CAR T and BCMAxCD3 TCE therapy and herein reported the first case of biallelic loss of GPRC5D post GPRC5DxCD3 TCE treatment. Importantly, we also identified a monoallelic loss of TNFRSF17 (del16p) coupled with BCMA extracellular domain point mutation [p.(R27P)] that confers resistance to two BCMAxCD3 TCEs in MM. Therefore, in addition to structural alterations (biallelic loss of TNFRSF17 or GPRC5D), antigen escape resulting from non-truncating missense point mutations in BCMA extracellular domain also mediate resistance to targeted-immunotherapies.

Identification of IL-4 As a Key Regulator of CAR T-Cell Exhaustion Using Functional Genomics and Correlates of the Zuma-1 Clinical Trial

Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CART19) has shown remarkable overall response rates in the treatment of hematological malignancies. However, durable response rates remain at approximately 40%. CART cell in vivo functions depend on their associated cell fate following infusion. T-cell exhaustion is an acquired and epigenetically regulated state of dysfunction that is associated with decreased proliferation and efficacy. While this phenomenon is widely considered a major limitation of CART cell therapy, mechanisms of CART cell exhaustion are poorly understood and vary depending on construct design and disease settings. Second generation CART19 cells with a CD28 costimulatory domain (CART19-28ζ) exhibit reduced persistence as compared to CART19 cells with a 4-1BB costimulatory domain. In this study, we aimed to interrogate the epigenetic landscape of exhaustion in CART19-28ζ cells. To ensure rigor, we employed the following three independent strategies: 1) RNA and ATAC sequencing on unstimulated vs. exhausted healthy donor CART19-28ζ cells by utilizing an in vitro model for exhaustion, 2) RNA and ATAC sequencing on pre-infusion axi-cel cell products from patients with B cell lymphoma treated in the pivotal Zuma-1 clinical trial that led to the FDA approval of axi-cel, comparing responders to non-responders, 3) a genome-wide CRISPR knockout screen in healthy donor CART19-28ζ cells. To begin our studies, we developed a 14-day in vitro repeated stimulation assay with healthy donor CART19-28ζ cells to model antigen-driven exhaustion. After 7 days of repeated stimulation through the CAR with the CD19+ tumor cell line, JeKo-1, CART19-28ζ cells demonstrated phenotypic and functional hallmarks of exhaustion as compared to baseline CART19-28ζ cells, as characterized by: 1) upregulated inhibitory receptors (PD-1: p <0.0001, TIM-3: p <0.0001, and LAG-3: p = 0.02), 2) decreased proliferation (p <0.0001), 3) CD4 loss (p < 0.0001), and 4) a decrease in the percent of cells with positive intracellular staining for stimulatory and effector cytokines (IL-2: p <0.0001 and TNF-α: p <0.0001). RNA sequencing identified 449 significantly upregulated genes and 320 significantly downregulated genes in exhausted CART19-28ζ cells. ATAC sequencing identified 411 gene regions with increased accessibility and 445 gene regions with decreased accessibility. Overlap of RNA and ATAC results showed 105 genes that were both differentially expressed and differentially accessible. Ingenuity pathway analysis of these genes showed activation of the T-cell exhaustion pathway (p = 0.004, Z= 1.0), a potential role for the Th1 and Th2 activation pathway (p < 0.0001) and identified IL-4 as a significant upstream regulator of exhaustion (p < 0.0001). Next, we studied the transcriptional and epigenetic signature of baseline axi-cel products by comparing responders, patients who achieved a complete remission as best response, to non-responders, patients who achieved stable or progressive disease as best response. RNA sequencing showed 54 differentially expressed genes and ATAC sequencing showed 24 differentially accessible gene regions. Only two genes, IL-4 and HLA-DQB1, were upregulated in non-responders based on both RNA and ATAC sequencing data (Figure 1A). Then, by using our in vitro exhaustion model to conduct a genome-wide CRISPR knockout screen in CART19-28ζ cells from healthy donors, we saw enrichment of genes involved in the suppression of IL-4- mediated signaling as identified by gene ontology enrichment analysis (p = 0.0001). Finally, to validate the functional impact of IL-4 on CART cells, we assessed effector functions of healthy donor CART19-28ζ cells following treatment with human recombinant IL-4. CART19-28ζ cells co-cultured with JeKo-1 in the presence of human recombinant IL-4 exhibited a reduction in antigen specific proliferation (p = 0.027), an increase in inhibitory receptor expression (TIM-3: p = 0.002 and LAG-3: p = 0.007), and a decrease in cytotoxicity as compared to vehicle treated cells (Figure 1B). IL-4 induced CART19-28ζ cell modulation was not due to a direct impact on tumor cells as identified by no change in tumor cytotoxicity or proliferation when JeKo-1 cells alone were treated with human recombinant IL-4. Together, this data suggests that the IL-4 axis may function as a key regulator of CART19 therapy failure associated with CART cell exhaustion. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD72 Is a Pan-Tumor Antigen Associated with Childhood Acute Leukemia

Immunotherapy has recently gained increasing attention as a new paradigm shift in cancer and has revolutionized the course of cancer treatment. The identification of ideal tumor-associated antigens (TAA) is a challenging process, since antigens have to be specific for the target cells and assure no off-target effects. We exploited a gene expression dataset of acute leukemias previously generated in our lab to identify potentially interesting TAAs. Among the most interesting hyper- and over-expressed genes with respect to normal bone marrow (BM) and with a surface protein localization, the CD72 was selected because of its specific association with childhood B-cell precursor acute lymphoblastic leukemias (BCP-ALL) and with a large proportion of pediatric acute myeloid leukemias (AML) cases. Based on the findings described below, we here propose CD72 as a novel and robust pan-acute leukemia TAA. Even though CD72 is known to be expressed by B-lineage cells, we profiled its expression in a variety of cell lines and primary samples, providing a map of its expression. CD72 was expressed by BCP-ALL cell lines (NALM-6, SEM, RS-4;11, RHC-ACV) and various AML cell lines (SHI-1, SKNO-1, MV4;11, MOML-13). Conversely, T-ALL cell lines (DND-41, TALL-1, RPMI-8402, ALL-SIL, JURKAT) and non-hematopoietic tumor cell lines (DAOY, NB-1, HT-29, A549, HeLa, MCF7) did not express the antigen. This supports the idea that CD72 is specific for hematopoietic tumors. Furthermore, we tested CD72 expression on major populations within normal BM specimens and on other healthy tissues: skin fibroblasts, mesenchymal stromal cells, skeletal myoblasts, endothelial cells. We confirmed that the antigen is expressed on B-lineage cells, whereas in other cell types we did not detect any signal. Then, to assess the interest of CD72 for the development of chimeric antigen receptor (CAR)-based immunotherapy approaches, we documented that CD72 was not expressed on CD34-positive/CD38-negative hematopoietic stem and progenitor cells (HSPCs) from both mobilized peripheral blood cells and healthy-regenerating BM samples (Figure 1). We profiled CD72 expression by flow-cytometry on 289 pathological pediatric BM samples, including: 93 BCP-ALL at diagnosis and 44 at relapse; 38 T-ALL at diagnosis and 3 at relapse; 68 AML at diagnosis and 12 at relapse; blast cells from 6 myelodysplastic syndromes; 14 non-hematopoietic solid tumors invading BM (4 rhabdomyosarcomas, 6 neuroblastomas, 4 Ewing's sarcomas); blast cells from 3 T-cell Lymphoblastic Lymphomas and 8 B-lineage Non-Hodgkin Lymphomas (NHL) with BM involvement. Data from clinical samples confirmed the in silico and in vitro prediction. CD72 was positive in 134/137 (98%) of the examined BCP-ALL cases and in all B-lineage NHL and 52/80 (65%) of AML cases. Conversely, CD72 was expressed only in 2/44 (4%) of T-ALL cases and was absent in T-cell NHL and in non-hematopoietic solid tumors. Notably, CD72 was expressed by all 12 BCP-ALL patients that were previously treated with CD19-directed immunotherapies, including four patients who lost CD19 expression. Moreover, CD72 was positive on two rare cases of CD19-negative BCP-ALL at diagnosis (Figure 2). Within our AML cohort, we did not detect any difference in CD72 expression across AML genetic subgroups. Taken together, the preliminary data here reported highlight the potential clinical usefulness of CD72 for advanced diagnostics, including the ability to identify blast cells in case of CD19 loss, and provide support for developing novel immunotherapy approach directed at CD72 that may be endowed with the unique potential of targeting both acute lymphoid and myeloid leukemia. Figure 1. CD72 expression by flow-cytometry on healthy CD34+ and CD34+/CD38- cells from healthy-regenerating bone marrow. CD72 expression was evaluated according to AIEOP-BFM Consensus guidelines, comparing the fluorescence shift and distribution pattern of the cells to the appropriate negative control. Figure 2. CD72 expression on CD19-negative BCP-ALL by flow-cytometry at first diagnosis and at time of relapse after CD19-directed immunotherapy. B-lineage was assigned with the contribution of other antigens relevant in immunophenotyping of acute leukemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal