Loss Of CD4 Research Articles

Abstract 3294: In vivo delivery of a novel CD3-targeted lentiviral vector generates CD19 CAR-T cells in two different humanized mouse models and results in complete B cell depletion

Abstract Introduction: Chimeric antigen receptor therapies (CAR-T) are highly effective against hematologic malignancies but require a lymphodepleting chemotherapy regimen and are faced with many challenges including manufacturing time, scalability, and cost of production due to the need for ex vivo culturing of cells and complex chain of custody requirements. In vivo delivery of self-inactivating lentiviral vectors (LV) encoding for CAR-T transgenes represents a promising strategy to improve the time to treatment, scalability, and cost of current CAR-T therapies. Methods: Lentiviral vectors encoding a CD19 CAR with a synthetic driver element were manufactured in a chemically defined cell substrate that incorporates a modified envelope designed to target and activate CD3+ T cells. Two different humanized mouse models were utilized. In a PBMC humanized mouse model, NSG MHC Class I/II knock-out mice (DKO) were injected with 1E7 human PBMC and followed one day later with 2E7 TU of LV encoding for CD19 CAR. In parallel, CD34+ humanized NSG SGM3 mice were also administered 2E7 TU LV encoding CD19 CAR. Flow cytometry of peripheral blood samples was evaluated at various time points for the presence of CAR+ cells and CD20+ B cells. Additionally, tissue samples were examined by histopathology and PCR for vector copy numbers. Results: The NSG SGM3 humanized CD34+ mouse model exhibited efficient chimerism of human CD45+ hematopoietic cells (>50% of live cells in peripheral blood). Apart from CD15+ neutrophils, all major human immune cell components were well represented in peripheral blood including CD14+ monocytes, CD20+ B cells, and CD3+ lymphocytes. At study initiation, the T cell compartment in the SGM3 mice exhibited skewing towards CD4+ T cells. Injection of CD19 CAR-LV resulted in a significant reduction of CD20+ B cells as early as 5 days post-injection. CD3+ CAR+ cells were detected in peripheral blood by day 5 with evidence of CAR+ cell expansion at subsequent time points. Loss of CD20+ B cells was stable throughout the observation period with some mice exhibiting a complete elimination of B cells. Similarly, the PBMC humanized NSG DKO mice injected with CD19 CAR-LV also showed evidence of CAR+ cells in peripheral blood. Conclusions: CD3-directed self-inactivating lentiviral vectors can efficiently deliver an integrating CAR gene into T lymphocytes following in vivo administration. These in vivo generated CD19 CAR T cells expand systemically and effectively eliminate pre-existing B cells. These data show that the targeting of CD3 through in vivo delivery can produce functional CAR T cells and represents an innovative therapeutic opportunity to potentially overcome current manufacturing times, scalability, and cost challenges facing cell therapies. Citation Format: Frederic Vigant, Ani Kundu, Dongming Zhang, Wei Zhang, Ewa Jaruga-Killeen, Michelle Andraza, Gregory Schreiber, Alissa Kerner, Junyi Zhang, John Henkelman, Renata Soares, Ramya Yarlagadda, Gregory I. Frost, Sid P. Kerkar. In vivo delivery of a novel CD3-targeted lentiviral vector generates CD19 CAR-T cells in two different humanized mouse models and results in complete B cell depletion [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 3294.

Abstract 1363: Preclinical development of bispecific CAR-T cells for canine B cell malignancies

Abstract Chimeric antigen receptor (CAR)-T cells targeting CD19 have revolutionized the treatment of B cell malignancies in people. However, post-treatment loss of CD19 has emerged as an adaptive mechanism of resistance. Pet dogs that develop tumors spontaneously in an immune competent setting represent a compelling parallel population of patients to study next generation cancer immunotherapies. Comparative trials at the University of Pennsylvania are investigating the activity of canine CAR-T cells targeting either CD19 or CD20 in canine B cell lymphoma patients and have revealed a favorable safety profile with early signs of anti-tumor activity. Aligned with experiences in human trials, expansion of CD20-negative B cells has been observed in dogs treated with anti-CD20 CAR-T cells. Bispecific CAR-T cells that simultaneously target two antigens to mitigate antigen escape have encouraging efficacy profiles in human patients. Thus, we designed a dual-targeting CAR specific for canine B cell antigens. A second-generation canine CAR construct targeting both CD19 and CD20 with a 28z signaling domain was cloned into the MSGV1 backbone. Chimeric retrovirus encoding RD114 and VSV-G envelope proteins and the CAR construct was made using GP2-293 cells. Jurkat NFAT-GFP reporter cells and primary T cells from dogs diagnosed with high grade B cell lymphoma were retrovirally transduced with bispecific CARs. Antigen specific reactivity was assessed against cell lines with variable CD19 and CD20 expression patterns. The CD19-CD20 CAR was efficiently transduced, with stable expression in both Jurkat and primary canine T cells. Jurkat reporters revealed dual-specific CAR signaling after co-culture with CD19+ and/or CD20+ target cells. Primary canine CAR-T cells expanded in vitro and exhibited CAR-specific proliferation and cytotoxicity against CD19+ CD20+ target cells. Preclinical manufacture of functional, bispecific anti-CD19 anti-CD20 canine CAR-T cells is feasible. A clinical trial recruiting dogs diagnosed with B cell cancer will assess safety and efficacy of this approach in vivo, with the aim of establishing the dog as a compelling model to appraise the ability of bispecific CAR-T cells to overcome therapeutic failures secondary to antigen loss. Citation Format: Matthew J. Atherton, Antonia Rotolo, Nicola J. Mason. Preclinical development of bispecific CAR-T cells for canine B cell malignancies [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 1363.

Characterization of CD20 expression loss as a mechanism of resistance to mosunetuzumab in patients with relapsed/refractory B-cell non-Hodgkin lymphomas.

7526 Background: Mosunetuzumab (M) is a bispecific antibody targeting CD20 and CD3 that redirects T cells to engage and eliminate malignant B cells being developed for relapsed or refractory (R/R) B-cell non-Hodgkin lymphomas (B-NHL). CD20 is an optimal target, with uniform expression across B-NHL histologies and minimal receptor turnover. We characterized CD20 loss as a potential mechanism of resistance to M in patients (pts) on a Phase I/II trial (NCT02500407) receiving M monotherapy for the treatment (tx) of R/R B-NHL. Methods: Pts with R/R B-NHL received M intravenously in 3-week cycles, for eight to 17 cycles depending on tumor response. At baseline (BL), biomarker-evaluable (archival or fresh) biopsies were collected from 293 pts. Biopsies from 62 pts were collected at additional time points during tx with M and/or at disease progression (PD). The proportion of CD20+ and PAX5+ tumor cells was determined by immunohistochemistry (IHC) using dual-staining with anti-CD20 (clone L26, VENTANA) and anti-PAX5 (clone DAK-PAX5, DAKO) antibodies. Expression of MS4A1, the gene encoding CD20 , was measured by RNA-sequencing (RNA-seq); MS4A1 mutation profiling was performed by whole exome sequencing (WES). Levels of CD20 expression were assessed relative to response rates. Correlative analyses were performed and assessed centrally (IHC, RNA-seq, and WES) and locally (IHC). Results: CD20 levels were consistently high ( > 75% CD20+PAX5+ cells) in the majority of BL biopsies and generally comparable across histologies (FL, DLBCL, tFL, MCL, and RT). BL CD20 loss (≤5% CD20+PAX5+ cells) was seen in 16/293 pts (5.5%), more commonly in aggressive NHL, and responses to M were not seen in these pts. Among 62 pts with BL and on-tx/at-PD biopsies, BL CD20 levels were ≤5% in 7/62 pts (11%) (6/7 pts [86%] progressed before completing Cycle 2). CD20 levels were maintained in on-tx biopsies from 23/24 pts (96%). At PD, biopsies showed CD20 loss in 7/26 pts (27%). For five pts with BL, on-tx and at-PD biopsies, all pts maintained CD20 while on-tx and 1/5 pts (20%) had CD20 loss at PD. There was no clear association between CD20 reduction and histology. Data from 185 BL biopsies showed generally concordant levels of CD20 gene and protein expression (r = 0.72). In 10/185 pts (5%), MS4A1 was expressed without detectable CD20 protein expression; DNA sequencing revealed novel mutations in MS4A1, including mutations leading to truncation of the protein. CD20 transmembrane and extra-cellular domain mutations were also observed but do not block CD20 expression. Conclusions: In pts with R/R B-NHL treated with M, low BL CD20 expression is associated with lack of response to M. During M tx, loss of tumor cell expression of CD20 is one mechanism of acquired resistance; however, CD20 expression is maintained in most pts with PD, implying alternative mechanisms for acquired M resistance. Clinical trial information: NCT02500407.

A real-world experience: Outcomes among relapsed/refractory diffuse large B cell lymphoma patients with CD20 loss.

e19570 Background: Despite novel therapies for relapsed/refractory (R/R) DLBCL, outcomes among this cohort remain poor. A prior study indicated that patients who lost CD20 expression after treatment have earlier disease progression, but whether this translates to inferior outcomes remains unknown. We hypothesized that R/R DLBCL patients who lost CD20 expression upon disease relapse would have worse survival and response rates compared to those who retained expression. Methods: We performed a retrospective cohort study of R/R DLBCL patients seen at the University of Michigan between 2007-2021. 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/immunohistochemistry. Overall survival (OS) was defined as time from first relapse to death and relapse-free survival (RFS) 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: 98 patients were included (65% male) with median age at diagnosis of 62 years. Majority of patients had stage III/IV disease at diagnosis (85%), 45% with GCB subtype and 7% with high-grade lymphoma. At diagnosis, all patients had CD20+ disease. Median time from diagnosis to first relapse was 7 months and median time from completion of induction treatment to first relapse was 2.4 months. Among the 98 patients, 84 were CD20+ at first relapse, 12 were CD20 negative and the remaining 2 patients were indeterminate and excluded from survival analyses. The estimated OS in the CD20+ group was 17.9 months (95% CI 14.0, 41.8) compared to 10.6 months in the CD20 negative group (95% CI 6.9, Not Reached). Although median OS in the CD20+ group was longer, a two-sample log-rank test failed to identify a significant difference in OS between the two groups (p = 0.085). The estimated RFS in the CD20+ group was 3.5 months (95% CI 3.3, 3.9) compared to 4.4 months in the CD20 negative group (95% CI 2.6, Not Reached, p = 0.739). ORR among CD20+ and CD20 negative patients was 64% and 50%, respectively. Of patients who became CD20 negative at relapse, 100% received anti-CD20 directed therapy thereafter, as part of salvage or with conditioning prior to transplant. Conclusions: Patients with DLBCL who relapse and maintain CD20 positivity seem to have a trend towards longer OS, though this was not statistically significant in our analysis. Our study was limited given the retrospective data collection and small sample size which may have limited our ability to detect a statistical significance in outcomes. In the future, we will plan to expand our study to involve other academic institutions and reassess outcomes among these cohorts.

CD20 and CD19 expression loss in relapsed or refractory b-cell non-Hodgkin lymphoma: A retrospective cohort.

e19537 Background: CD20-targeted therapies such as rituximab are widely used in the management of B-cell non-Hodgkin lymphoma (NHL). Re-treatment with anti-CD20 agents is common, however previous research has demonstrated loss of CD20 expression at time of relapse or refractory disease in a subset of patients. There is also amplified interest in alternative targets, specifically CD19, in the relapsed or refractory setting. Incidence of CD19 loss has been explored in post-CAR T cell therapy patients but not described elsewhere. We hypothesized that CD marker loss may be higher than previous reports due to expanded use of CD20- and CD19-targeted therapy. Methods: The present study is a retrospective cohort analysis of 229 patients with B-cell NHL from Harbor-UCLA Medical Center in Los Angeles, CA from 2006 to 2022. Charts were retrospectively reviewed for cases of B-cell NHL, and data was collected about demographics, diagnosis, treatment, and relapses. CD20 and CD19 expression analysis was performed by immunohistochemistry and / or flow cytometry at diagnosis and at each relapse whenever possible. The primary endpoint of the study was the rate of CD marker expression loss at time of relapse. Results: The cohort of 229 patients was composed of 54.1% diffuse large B-cell lymphoma (DLBCL), 14.4% follicular lymphoma (FL), 8.3% high-grade B-cell lymphoma (HGBCL), 7.0% marginal zone lymphoma (MZL), 4.8% mantle cell lymphoma (MCL), and 11.2% other. Overall, 28.8% (66/229) patients had relapsed or refractory disease. The rate of CD20 expression loss was 7.9% (5/63) across all relapses with two patients converting at first relapse (1 FL relapsing as HGBCL, 1 MZL), two patients at 2nd relapse (1 FL relapsing as DLBCL, 1 MZL relapsing as DLBCL), and one patient at 5th relapse (1 MZL). All five patients with CD20 phenotypic conversion received rituximab, and three patients received two or more rounds of anti-CD20 therapy. The median OS after CD20 expression loss was 4 months (95% CI 3.3 – 4.7 months). All patients (14/14) who had CD19 testing performed at relapse maintained CD19 positivity. Conclusions: This retrospective study demonstrates a rate of CD20 expression loss at relapse of 7.9%, which is less than previously reported rates ranging between 11-60%. CD20 expression loss seems to be associated with transformation to high-grade lymphoma, poor overall survival, and marginal zone histology. The conservation of CD19 expression across relapses supports the use of novel anti-CD19 targeted therapy in the relapsed or refractory setting. Whenever possible, repeat biopsy should be done at time of relapse prior to treatment with CD-targeted therapy. This study is of notable clinical relevance with expanded use of anti-CD20 agents and novel CD19-targeted therapies in the management of B-cell NHL.

CAR-T Cells Shoot for New Targets: Novel Approaches to Boost Adoptive Cell Therapy for B Cell-Derived Malignancies.

Chimeric antigen receptor (CAR)-T cell therapy is undeniably a promising tool in combating various types of hematological malignancies. However, it is not yet optimal and a significant number of patients experience a lack of response or relapse after the treatment. Therapy improvement requires careful analysis of the occurring problems and a deeper understanding of the reasons that stand behind them. In this review, we summarize the recent knowledge about CAR-T products’ clinical performance and discuss diversified approaches taken to improve the major shortcomings of this therapy. Especially, we prioritize the challenges faced by CD19 CAR-T cell-based treatment of B cell-derived malignancies and revise the latest insights about mechanisms mediating therapy resistance. Since the loss of CD19 is one of the major obstacles to the success of CAR-T cell therapy, we present antigens that could be alternatively used for the treatment of various types of B cell-derived cancers.

Myelomatous pleural effusion.

A 66-year-old woman was admitted to the emergency department reporting 1 week of asthenia and fever that had persisted despite antibiotherapy. She had been diagnosed of smoldering multiple myeloma (MM) IgG kappa 11 years before. However, treatment with bortezomib, lenalidomide and dexamethasone (VRd) was not started until the previous year, when the patient presented with rib pain and the diagnostic workup was repeated. Bone marrow aspirate showed infiltration of clonal plasma cells, and bone disease was demonstrated in positron emission tomography/computed tomography scan. After the fourth cycle of VRd, an increase in serum monoclonal component was observed. Urgent laboratory tests showed hemoglobin, white blood cell and platelet counts of 110 g/L, 7.5 × 109/L and 190 × 109/L, respectively, with 1570 mg/dl of IgG, 643 mg/dl of kappa light chain, 933 U/L of LDH, and 6.5 mg/L of beta2-microglobulin. Chest X-ray revealed left pleural effusion (PE). A thoracocenthesis was performed. Cytological examination of PE and peripheral blood smear showed plasma cell infiltration (Figure 1A–F). PE flow cytometry revealed 96% infiltration of abnormal cells expressing CD38, CD138, CD56dim and CD19 loss expression (Figure 2A–E). Pleural fluid electrophoresis and immunofixation revealed monoclonal IgG kappa (Figure 2F–G) and bone marrow aspirate showed 90% infiltration of plasma cells. Blood and bone marrow cultures were negative. A diagnosis of myelomatous pleural effusion (MPE) was made. Treatment with daratumumab, carfilzomib and dexamethasone was started. However, 10 days later the patient died. MM is a malignant plasma cell disorder that accounts for approximately 10% of all hematologic malignancies. Only 6% of these patients develop pleural effusion, mostly benign [1]. MPE is a very rare finding, present in less than 1% of all cases. Cytological identification of malignant plasma cells within pleural effusion is considered the most accurate diagnostic method. There is no standardized therapy. The prognosis is dismal, with an overall median survival ranging from 2 to 4 months [2-4]. None. The authors declare no conflict of interest. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from a first degree relative of the patient included in the study. All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Silvia Escribano Serrat, Javier Cucharero Martín, Fiorella Medina Salazar, Estefanía Bolaños Calderón, Isabel Ortega Madueño and Fernando Ataúlfo González Fernández. The first draft of the manuscript was written by Silvia Escribano Serrat and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. None.

CD4/CD8 double-negative mycosis fungoides with large cell transformation and involvement of the lungs and leptomeninges.

Mycosis fungoides (MF), the most common cutaneous T-cell lymphoma, classically has an indolent clinical course, with lesions slowly progressing from patch to plaque to tumor stage. In some cases, the late stages of disease involve extra-cutaneous dissemination to lymph nodes or viscera. Although this "Alibert-Bazin" type is the prototypic MF, there are several variants and subtypes of MF that may have different clinical implications for treatment and prognosis. We describe a woman whose disease course involved a variety of histopathologic and immunophenotypic variants including folliculotropic MF, granulomatous MF with loss of CD8, and then finally CD4/CD8 double-negative MF with large cell transformation and extra-cutaneous dissemination. Clinically her disease behaved as classic indolent stage IA MF for nearly two decades before transitioning to tumor stage and then, finally, involving the lungs and leptomeninges. It is important for physicians to be aware of the clinically relevant variants of MF as well as the possibility of transformation of previously stable disease both clinically and histopathologically.

Prevalence of T-cell antigen losses in mycosis fungoides and CD30-positive cutaneous T-cell lymphoproliferations in a series of 153 patients

Mycosis fungoides (MF) and primary cutaneous CD30-positive T-cell lymphoproliferative disorders (CD30LPD) are the most frequent primary cutaneous T-cell lymphomas. Our objective was to study pan-T-cell antigens and PD-1 expression in a large cohort of MF and CD30LPD with a special interest in antigen losses as a diagnostic tool. We retrospectively reviewed 160 consecutive samples from 153 patients over a 3 year period, including 104 with MF and 49 with CD30LPD. As controls, benign inflammatory dermatoses (BID, n=19) were studied. A semi-quantitative evaluation of CD2, CD3, CD4, CD5, CD7, CD8 expression was performed. PD-1 and double stainings (CD3+CD7 and PD-1+CD7) were performed in a subset of MF cases. CD8+ MF was frequent (23%) and CD7 was the most frequently lost antigen in both MF (45%) and CD30LPD (86%), while no significant T-cell antigen loss was observed in BID. CD7 loss was less frequent in folliculotropic MF (p<0.001). PD-1 was variably expressed in MF with no differences with BID. The CD3+/CD7- and PD-1+/CD7- neoplastic lymphocytes were highlighted by the use of chromogenic double staining experiments in MF with a CD7 loss identified with single staining. Multiple pan T-cell antigen losses were mostly seen in CD30LPD with CD2 being the most frequently preserved marker (90%). While PD-1 does not discriminate between MF and BID, CD7 is frequently lost in MF infiltrates as well as other pan-T-cell antigens in CD30LPD, which can be used as routine markers for diagnosis. We recommend the use of CD7 in addition to CD3, CD4 and CD8 as a minimal immunohistochemical panel for MF assessment, and the use of double stainings for CD3 and CD7 in difficult cases.

CD4 Tcells are rapidly depleted from tuberculosis granulomas following acute SIV co-infection.

HIV/Mycobacterium tuberculosis (Mtb) co-infected individuals have an increased risk of tuberculosis prior to loss of peripheral CD4 Tcells, raising the possibility that HIV co-infection leads to CD4 Tcell depletion in lung tissue before it is evident in blood. Here, we use rhesus macaques to study the early effects of simian immunodeficiency virus (SIV) co-infection on pulmonary granulomas. Two weeks after SIV inoculation of Mtb-infected macaques, Mtb-specific CD4 Tcells are dramatically depleted from granulomas, before CD4 Tcell loss in blood, airways, and lymph nodes, or increases in bacterial loads or radiographic evidence of disease. Spatially, CD4 Tcells are preferentially depleted from the granuloma core and cuff relative to B cell-rich regions. Moreover, live imaging of granuloma explants show that intralesional CD4 Tcell motility is reduced after SIV co-infection. Thus, granuloma CD4 Tcells may be decimated before many co-infected individuals experience the first symptoms of acute HIV infection.

Induction of Autophagy reduces IFN-I mediated Inflammation and restores anti-HIV-1 T Cell response in vivo

Abstract A hallmark of HIV-1 infection is chronic inflammation. Chronic immune activation drives the pathogenesis of HIV-1 infection, leading to loss of CD4+ T cells and immune exhaustion. Previously, we demonstrated that persistent type I interferon (IFN-I) signaling drives T cell exhaustion during chronic HIV infection and combined anti-retroviral therapy (ART) and IFN-I receptor blockade could lead to reduced inflammation, accelerated viral suppression and HIV reservoir. However, as IFN-Is are key regulators for antiviral immunity, more specific interventions to fine-tune IFN-I signaling and chronic inflammation are needed. Autophagy is a homeostatic mechanism for disposal of damaged cellular organelles and elimination of intracellular pathogens, which is pivotal for cellular homeostasis, T cell development and function. Autophagy is also impaired during HIV-1 infection. Here we demonstrate that autophagy is directly linked to IFN-I signaling. Impairment of autophagy leads to accumulation of damaged mitochondria and spontaneous IFN-I signaling. Autophagy inducers reduce IFN-I signaling in activated macrophages and restore functions of exhausted anti-HIV-1 T cells in vitro. Importantly, autophagy inducer treatment in HIV-1 infected humanized bone marrow/liver/thymus (BLT) mice significantly reduced persistent IFN-I signaling and immune activation, restored exhausted antiviral T cell responses, and accelerated viral suppression by ART. Autophagy inducer treatment also led to reduced viral rebound after ART withdrawal. Taken together, our data suggest that therapeutically targeting autophagy is a promising approach for treating persistent immune activation and improve immune control of HIV replication. This work was funded by NIAID 1R21AI140866 (to Zhen), NIDA R01DA-52841 (to Zhen), NIAID R2120200174 (PIs: Xie&amp;Zhen), NCI 1R01CA239261-01 (to Kitchen), NIH Grants NIH Grants P30AI28697 (the UCLA CFAR Virology Core, Gene and Cell Therapy Core, and Humanized Mouse Core), U19AI149504(PIs: Kitchen &amp; Chen), CIRM DISC2-10748, UPLIFT: UCLA Postdocs’ Longitudinal Investment in Faculty (Award # K12 GM106996) (to Carrillo), R01 AG052340 (to Jamieson). This work was also supported by the UCLA AIDS Institute, the James B. Pendleton Charitable Trust, and the McCarthy Family Foundation. We would also like to thank Drs. Romas Geleziunas and Jeff Murry and the people at Gilead for providing the antiretroviral drugs used in this study.

Whole-genome sequencing reveals complex genomic features underlying anti-CD19 CAR T-cell treatment failures in lymphoma

AbstractCD19-directed chimeric antigen receptor (CAR-19) T cells are groundbreaking immunotherapies approved for use against large B-cell lymphomas. Although host inflammatory and tumor microenvironmental markers associate with efficacy and resistance, the tumor-intrinsic alterations underlying these phenomena remain undefined. CD19 mutations associate with resistance but are uncommon, and most patients with relapsed disease retain expression of the wild-type receptor, implicating other genomic mechanisms. We therefore leveraged the comprehensive resolution of whole-genome sequencing to assess 51 tumor samples from 49 patients with CAR-19–treated large B-cell lymphoma. We found that the pretreatment presence of complex structural variants, APOBEC mutational signatures, and genomic damage from reactive oxygen species predict CAR-19 resistance. In addition, the recurrent 3p21.31 chromosomal deletion containing the RHOA tumor suppressor was strongly enriched in patients for whom CAR T-cell therapy failed. Pretreatment reduced expression or monoallelic loss of CD19 did not affect responses, suggesting CAR-19 therapy success and resistance are related to multiple mechanisms. Our study showed that tumor-intrinsic genomic alterations are key among the complex interplay of factors that underlie CAR-19 efficacy and resistance for large B-cell lymphomas.

Clinical and Clinical Pathological Presentation of 310 Dogs Affected by Lymphoma with Aberrant Antigen Expression Identified via Flow Cytometry.

Phenotypic aberrancies have been reported occasionally in canine lymphomas. Here, we retrospectively collected 310 canine lymphomas with an aberrant phenotype detected via flow cytometry and describe their clinical and clinical pathological features at diagnosis. There were 152 T-cell lymphomas not otherwise specified (T-NOS), 101 T-zone lymphomas (TZL), 54 B-cell lymphomas, and 3 cases with two suspected concurrent neoplastic populations. The most represented aberrancies were: CD5-, CD4-CD8-, and CD3- in T-NOS lymphomas, CD21+, CD4-CD8-, and CD3- in TZLs, and CD34+, CD44-, and CD5+ in B-cell lymphomas. Among T-cell lymphomas, the aberrant expression of CD21 was significantly more frequent in TZL and the loss of CD5 and CD44 in T-NOS. More than 75% of dogs were purebred; males outnumbered females; the mean age at diagnosis was 8–10 years, depending on lymphoma subtype. A few dogs were symptomatic at the time of diagnosis, and 30% had peripheral blood abnormalities, in line with what is already reported for the general population of dogs with lymphoma. Further studies are needed to assess the pathogenetic mechanisms underlying each specific antigen aberrancy, as well as the diagnostic and prognostic role.

Synapse topology and downmodulation events determine the functional outcome of anti-CD19 T cell-redirecting strategies

ABSTRACT Cancer immunotherapy strategies based on the endogenous secretion of T cell-redirecting bispecific antibodies by engineered T lymphocytes (STAb-T) are emerging as alternative or complementary approaches to those based on chimeric antigen receptors (CAR-T). The antitumor efficacy of bispecific anti-CD19 × anti-CD3 (CD19×CD3) T cell engager (BiTE)-secreting STAb-T cells has been demonstrated in several mouse models of B-cell acute leukemia. Here, we have investigated the spatial topology and downstream signaling of the artificial immunological synapses (IS) that are formed by CAR-T or STAb-T cells. Upon interaction with CD19-positive target cells, STAb-T cells form IS with structure and signal transduction, which more closely resemble those of physiological cognate IS, compared to IS formed by CAR-T cells expressing a second-generation CAR bearing the same CD19-single-chain variable fragment. Importantly, while CD3 is maintained at detectable levels on the surface of STAb-T cells, indicating sustained activation mediated by the secreted BiTE, the anti-CD19 CAR was rapidly downmodulated, which correlated with a more transient downstream signaling. Furthermore, CAR-T cells, but not STAb-T cells, provoke an acute loss of CD19 in target cells. Such differences might represent advantages of the STAb-T strategy over the CAR-T approach and should be carefully considered in order to develop more effective and safer treatments for hematological malignancies.

A novel and efficient CD22 CAR-T therapy induced a robust antitumor effect in relapsed/refractory leukemia patients when combined with CD19 CAR-T treatment as a sequential therapy

BackgroundCD19 chimeric antigen receptor (CAR) therapy has achieved impressive success in relapsed or refractory (R/R) B-cell malignancies, but relapse due to antigen escape is increasingly appearing reported. As the expression profile of CD22 is similar to that of CD19, CD22 has become a candidate target when CD19 CAR-T therapy fails.MethodsA novel CD22 CAR incorporating scFv derived from an HIB22 hybridoma which bound the first and second Ig-like extracellular domains of CD22 antigen was constructed. Preclinical investigation of the CD22 CAR-T therapy against B-cell malignancies was evaluated by coculturing CD22 CAR-T cells with tumor cell lines or primary blasts from patients in vitro and using a xenograft mouse model in vivo. Further clinical study of CD22/CD19 CAR-T sequential therapy was conducted in 4 R/R adult B-cell acute lymphoblastic leukemia (B-ALL) patients.ResultsThe novel CD22 CAR-T treatment had specific cytotoxicity to CD22 + target cells, and the survival time of mice in the CD22 CAR-T treatment group was significantly prolonged. Furthermore, it’s validated that sequential CD22/CD19 CAR-T therapy was significantly superior than single CD19 or CD22 CAR-T treatment in a relapse xenograft model. All 4 patients achieved complete remission (CR) with negative minimal residual disease (MRD), including 3 patients who had received prior CD19-related immunotherapy. The proliferation of CD19 and CD22 CAR-T cells was observed respectively in vivo, and 3 of the 4 patients experienced cytokine release syndrome (CRS); 2 of these patients had grade 1 CRS and 1 had grade 3 CRS. Long term follow-up showed that 3 of the 4 (75%) patients had sustained CR for up to 1 year. Analysis of antigen expression in the relapsed patients demonstrated that loss or diminution of CD19 and CD22 expression might cause antigen escape from CAR-T surveillance.ConclusionsIn summary, the novel CD22 CAR-T therapy was validated with antitumor effects both in vitro and in vivo. Furthermore, our study demonstrated the safety and robust efficacy of sequential CD22/CD19 CAR-T therapy in xenograft models and clinical trials, especially as the salvage treatment for R/R B-ALL patients with antigen loss or in whom anti-CD19 related immunotherapy failure failed.Trial registration: Chinese Clinical Trial Registry (ChiCTR): ChiCTR1900025419, Supplementarily registered 26 August, 2019.

CD5 Deficiency Alters Helper T Cell Metabolic Function and Shifts the Systemic Metabolome.

Metabolic function plays a key role in immune cell activation, destruction of foreign pathogens, and memory cell generation. As T cells are activated, their metabolic profile is significantly changed due to signaling cascades mediated by the T cell receptor (TCR) and co-receptors found on their surface. CD5 is a T cell co-receptor that regulates thymocyte selection and peripheral T cell activation. The removal of CD5 enhances T cell activation and proliferation, but how this is accomplished is not well understood. We examined how CD5 specifically affects CD4+ T cell metabolic function and systemic metabolome by analyzing serum and T cell metabolites from CD5WT and CD5KO mice. We found that CD5 removal depletes certain serum metabolites, and CD5KO T cells have higher levels of several metabolites. Transcriptomic analysis identified several upregulated metabolic genes in CD5KO T cells. Bioinformatic analysis identified glycolysis and the TCA cycle as metabolic pathways promoted by CD5 removal. Functional metabolic analysis demonstrated that CD5KO T cells have higher oxygen consumption rates (OCR) and higher extracellular acidification rates (ECAR). Together, these findings suggest that the loss of CD5 is linked to CD4+ T cell metabolism changes in metabolic gene expression and metabolite concentration.

Comparative assessment of immunological tolerance in fish with natural immunodeficiency

Natural occurrences of immunodeficiency by definition should lead to compromised immune function. The major histocompatibility complexes (MHC) are key components of the vertebrate adaptive immune system, charged with mediating allorecognition and antigen presentation functions. To this end, the genomic loss of the MHC II pathway in Syngnathus pipefishes raises questions regarding their immunological vigilance and allorecognition capabilities. Utilising allograft and autograft fin-transplants, we compared the allorecognition immune responses of two pipefish species, with (Nerophis ophidion) and without (Syngnathus typhle) a functional MHC II. Transcriptome-wide assessments explored the immunological tolerance and potential compensatory measures occupying the role of the absent MHC II. Visual observations suggested a more acute rejection response in N. ophidion allografts compared with S. typhle allografts. Differentially expressed genes involved in innate immunity, angiogenesis and tissue recovery were identified among transplantees. The intriguing upregulation of the cytotoxic T-cell implicated gzma in S. typhle allografts, suggests a prominent MHC I related response, which may compensate for the MHC II and CD4 loss. MHC I related downregulation in N. ophidion autografts hints at an immunological tolerance related reaction. These findings may indicate alternative measures evolved to cope with the MHC II genomic loss enabling the maintenance of appropriate tolerance levels. This study provides intriguing insights into the immune and tissue recovery mechanisms associated with syngnathid transplantation, and can be a useful reference for future studies focusing on transplantation transcriptomics in non-model systems.

Evaluation of cytokine levels in response to mitogen among HIV-1-infected blood cells and their relationships to the number of T cells

BackgroundHuman immunodeficiency virus-1 (HIV-1) infection causes loss and anergy of CD4+ and CD8+ T cells, leading to opportunistic infections, including tuberculosis (TB). QuantiFERON®-TB (QFT) is used as a diagnostic tool to detect TB, but it exhibits limited accuracy among subjects with low CD4+ T cell numbers, including HIV-1-infected individuals. The present study aimed to determine the effect of HIV-1 infection and patients’ blood T cell numbers on cytokine production in response to mitogen (Mit) stimulation. MethodsThe number of CD4+ and CD8+ T cells in HIV-1-infected individuals was quantified. Levels of various cytokines in Mit-stimulated and un-stimulated (Nil) supernatants of QFT gold “in tube” were assessed using a MAGPIX System. The correlation between cytokine levels and CD4+/CD8+ T cell counts in response to Mit was analyzed. The cytokine levels were compared between HIV-1-infected and healthy subjects. ResultsHIV-1-infected individuals (110) and control subjects (27) were enrolled. Interferon (IFN)-γ, interleukin-1 receptor antagonist (IL-1RA), IL-6, IL-8, and regulated on activation, normal T cell expressed and secreted (RANTES) values in Mit-Nil tubes showed a significant correlation with CD4+ T cell counts, while IFN-γ, IL-6, and IFN-γ-induced protein 10 (IP-10) values in Mit-Nil tubes had significant correlation with CD8+ T cell counts. IL-1RA, IL-8, IP-10, platelet-derived growth factor (PDGF)-BB, and RANTES levels in Nil tubes were significantly higher in the HIV-1-infected group. IFN-γ, IL-2, IL-5, IL-6, IP-10, and macrophage inflammatory protein-1β values in Mit-Nil tubes were significantly higher, and PDGF-BB and RANTES levels were significantly lower in the HIV-1-infected group. ConclusionThe functions of HIV-1-infected T cells and uninfected T cells, such as spontaneous and responsive cytokine production in response to Mit, were different. Our findings may be useful for developing new clinical tools for patients with low T cell counts. Additionally, the study provides new insights into the pathogenesis of HIV-1 infection.

Distinct Plasma Concentrations of Acyl-CoA-Binding Protein (ACBP) in HIV Progressors and Elite Controllers.

HIV elite controllers (ECs) are characterized by the spontaneous control of viral replication, and by metabolic and autophagic profiles which favor anti-HIV CD4 and CD8 T-cell responses. Extracellular acyl coenzyme A binding protein (ACBP) acts as a feedback inhibitor of autophagy. Herein, we assessed the circulating ACBP levels in ECs, compared to people living with HIV (PLWH) receiving antiretroviral therapy (ART) or not. We found lower ACBP levels in ECs compared to ART-naïve or ART-treated PLWH (p < 0.01 for both comparisons), independently of age and sex. ACBP levels were similar in ECs and HIV-uninfected controls. The expression of the protective HLA alleles HLA-B*27, *57, or *58 did not influence ACBP levels in ECs. ACBP levels were not associated with CD4 or CD8 T-cell counts, CD4 loss over time, inflammatory cytokines, or anti-CMV IgG titers in ECs. In ART-treated PLWH, ACBP levels were correlated with interleukin (IL)-1β levels, but not with other inflammatory cytokines such as IL-6, IL-8, IL-32, or TNF-α. In conclusion, ECs are characterized by low ACBP plasma levels compared to ART-naïve or ART-treated PLWH. As autophagy is key to anti-HIV CD4 and CD8 T-cell responses, the ACBP pathway constitutes an interesting target in HIV cure strategies.

CD19 or CD20 CAR T Cell Therapy Demonstrates Durable Antitumor Efficacy in Patients with Central Nervous System Lymphoma.

Patients with central nervous system (CNS) lymphomas have a poor prognosis. Chimeric antigen receptor-modified (CAR) T cells have shown remarkable efficacy for B-cell non-Hodgkin's lymphoma. However, few studies have reported the effects of CAR T cells in the treatment of CNS lymphoma, and the duration of remission is short. In this study, seven CNS lymphoma patients (six patients with secondary CNS lymphomas and one patient with primary CNS lymphoma) were treated with CD19 or CD20 CAR T cell therapy, and the clinical efficacy and toxicity profiles were evaluated. All patients responded to CAR T cell therapy. Four patients achieved complete remission (CR), while three demonstrated partial remission. We also found that either CD19 or CD20 CAR T cells could be detected in the cerebrospinal fluid of four patients. The median progression-free survival and median overall survival were not assessed. The median duration of CR was 22.4 months. Five patients (5/7) in this cohort received combination therapy (bridging with autologous hematopoietic stem cell transplantation and programmed death-1 inhibitor for maintenance treatment) and three (3/7) received CAR T cell therapy as soon as possible after the relapse of CNS lymphoma. This could help explain why these patients achieved long-term remission. After a median follow-up of 10.4 months, three patients demonstrated disease progression, and the antigen loss of CD20 was confirmed as the reason for relapse in one patient. The results of this study suggest that CD19 or CD20 CAR T cells are effective against CNS lymphoma. This research was registered at http://www.chictr.org.cn (No. ChiCTR2000036350).