Loss Of CD4 Research Articles

CD19 regulates innate immunity by the toll-like receptor RP105 signaling in B lymphocytes

AbstractLipopolysaccharide (LPS) is a major gramnegative bacterial component that stimulates innate immune response and also induces B-lymphocyte activation. Recent studies have revealed that common molecular patterns of microorganisms such as LPS are recognized by toll-like receptors (TLRs). B cells have 2 known TLRs that mediate LPS signaling, TLR4 and RP105 (CD180). While TLR4 is expressed on immune cells of various types, RP105 is preferentially expressed on mature B cells. Here we demonstrate that CD19 plays a major role in regulating signal transduction through RP105. Anti-RP105 ligation induced normal proliferation of B cells from mice deficient for MyD88, an adaptor protein that mediates most TLR pathways. By contrast, the loss of CD19 resulted in modest B-cell proliferation against anti-RP105 stimulation as well as LPS stimulation. LPS induced tyrosine phosphorylation of CD19, which was RP105-dependent but TLR4-independent. CD19 formed a complex with Lyn and Vav following RP105 ligation, and CD19 expression was required for optimal Lyn activation and Vav phosphorylation. Consistently, B cells deficient for CD19 exhibited specific defect in the activation of c-Jun N-terminal kinases following RP105 ligation and LPS stimulation. In contrast, CD19 and phosphatidylinositol 3-kinase independently regulated intracellular calcium mobilization induced by anti-RP105 stimulation. Thus, signaling through the B-cell–specific LPS receptor RP105 is uniquely regulated by the B-cell–specific signaling component, Lyn/CD19/Vav complex.

Alloy CsCd x Pb1-x Br3 Perovskite Nanocrystals: The Role of Surface Passivation in Preserving Composition and Blue Emission.

Various strategies have been proposed to engineer the band gap of metal halide perovskite nanocrystals (NCs) while preserving their structure and composition and thus ensuring spectral stability of the emission color. An aspect that has only been marginally investigated is how the type of surface passivation influences the structural/color stability of AMX3 perovskite NCs composed of two different M2+ cations. Here, we report the synthesis of blue-emitting Cs-oleate capped CsCdxPb1–xBr3 NCs, which exhibit a cubic perovskite phase containing Cd-rich domains of Ruddlesden–Popper phases (RP phases). The RP domains spontaneously transform into pure orthorhombic perovskite ones upon NC aging, and the emission color of the NCs shifts from blue to green over days. On the other hand, postsynthesis ligand exchange with various Cs-carboxylate or ammonium bromide salts, right after NC synthesis, provides monocrystalline NCs with cubic phase, highlighting the metastability of RP domains. When NCs are treated with Cs-carboxylates (including Cs-oleate), most of the Cd2+ ions are expelled from NCs upon aging, and the NCs phase evolves from cubic to orthorhombic and their emission color changes from blue to green. Instead, when NCs are coated with ammonium bromides, the loss of Cd2+ ions is suppressed and the NCs tend to retain their blue emission (both in colloidal dispersions and in electroluminescent devices), as well as their cubic phase, over time. The improved compositional and structural stability in the latter cases is ascribed to the saturation of surface vacancies, which may act as channels for the expulsion of Cd2+ ions from NCs.

HIV Skews a Balanced Mtb-Specific Th17 Response in Latent Tuberculosis Subjects to a Pro-inflammatory Profile Independent of Viral Load

HIV infection predisposes latent tuberculosis-infected (LTBI) subjects to active TB. This study is designed to determine whether HIV infection of LTBI subjects compromises the balanced Mycobacterium tuberculosis (Mtb)-specific T helper 17 (Th17) response of recognized importance in anti-TB immunity. Comparative analysis of Mtb- and cytomegalovirus (CMV)-specific CD4+ Tcell responses demonstrates a marked dampening of the Mtb-specific CD4+ Tcell effectors and polyfunctional cells while preserving CMV-specific response. Additionally, HIV skews the Mtb-specific Th17 response in chronic HIV-infected LTBI progressors, but not long-term non-progressors (LTNPs), with preservation of pro-inflammatory interferon (IFN)-γ+/interleukin-17+ (IL-17+) and significant loss of anti-inflammatory IL-10+/IL-17+ effectors that is restored by anti-retroviral therapy (ART). HIV-driven impairment of Mtb-specific response cannot be attributed to preferential infection as cell-associated HIV DNA and HIV RNA reveal equivalent viral burden in CD4+ Tcells from different antigen specificities. We therefore propose that beyond HIV-induced loss of Mtb-specific CD4+ Tcells, the associated dysregulation of Mtb-specific Tcell homeostasis can potentially enhance the onset of TB in LTBI subjects.

Bronchiolitis obliterans syndrome is associated with increased senescent lymphocytes in the small airways

Immunosuppression therapy is ineffective at preventing bronchiolitis obliterans syndrome (BOS), primarily a disease of the small airways (SAs). Our previous reports show increased senescent CD28null T and natural killer T (NKT)-like cells in the peripheral blood of patients with BOS and increased cytotoxic, proinflammatory lymphocytes in the SAs. We hypothesized that the cytotoxic, proinflammatory lymphocytes in the SAs would be steroid-resistant senescent CD28null lymphocytes. Intracellular cytotoxic mediator granzyme B, interferon (IFN)-γ and tumor necrosis factor (TNF)-α proinflammatory cytokines, and CD28 were measured in the blood, bronchoalveolar lavage, large airway, and SA brushing T and NKT-like cells from 10 patients with BOS, 11 stable lung transplant recipients, and 10 healthy age-matched controls. SA brushings were cultured in the presence of ±1 µmol/liter prednisolone, ±5 mg/liter theophylline, and ±2.5 ng/ml cyclosporine A, and IFN-γ and TNF-α proinflammatory cytokines were assessed using flow cytometry. Increased SA CD28null T and NKT-like cells were identified in patients with BOS compared with that in the controls and stable transplant recipients. Loss of CD28 was associated with increased T and NKT-like cells expressing granzyme B, IFN-γ, and TNF-α. Loss of CD28 expression by CD8+ T cells was significantly associated with forced expiratory volume in 1 sec (R = 0.655, p = 0.006) and with time after transplantation (R = -0.552, p = 0.041). Treatment with prednisolone + theophylline + cyclosporin A inhibited IFN-γ and TNF-α production by SA CD28null CD8+ T and NKT-like cells additively. BOS is associated with the loss of CD28 in SA cytotoxic, proinflammatory senescent T and NKT-like lymphocytes. Treatment options that target the proinflammatory nature of these cells in the SAs may improve graft survival.

Hypopigmented Mycosis Fungoides: A Clinical and Histopathology Analysis in 9 Children.

Hypopigmented mycosis fungoides (HMF) is an uncommon variant of mycosis fungoides. To study the clinical and histopathology presentation in children with HMF. We reviewed 9 children diagnosed with HMF. The clinical data were collected and analyzed. Eight boys and 1 girl were included, with a median onset age of 7.4 year old and median age of diagnosis of 10.5 year old. Multiple hypopigmented patches were observed in all patients, and 5 patients exhibited multiple scaly erythema at the center of hypopigmented patches. Histopathology showed atypical lymphocytes with hyperchromatic, irregular, and cerebriform nuclei, infiltrated in the epidermis and dermis. Pautrier's microabscesses was noted in 6 of 9 patients, and papillary dermal fibroplasia was noted in 6 of 9 patients. CD8 predominance was detected in 4 of 6 patients. Four patients were simultaneously subjected to skin biopsy on hypopigmented patches and scaly erythema simultaneously. Compared with hypopigmented specimens, erythema biopsy detected deeper and denser infiltration of atypical lymphoid cells in 3 of 4 patients, higher CD4+/CD8+ ratio in 4 of 4 patients, more CD5 loss in 2 of 4 patients, and more CD7 loss in 2 of 4 patients. TCR gene monoclonal rearrangement was detected in 2 of 5 patients. Narrowband ultraviolet B phototherapy was applied in 7 patients. One of 7 patients achieved complete response, and 6 of 7 patients achieved partial response. No recurrence was noted with the median follow-up period of 6 months. HMF could occur in young patients, with indolent and benign course. HMF could gradually seem as scaly erythema based on hypopigmented patches. The histopathology indicated a more advanced stage of the scaly erythema lesions than hypopigmented patches.

Detailed Immunophenotypic Profiling of Peripheral Blood Immune Cells in Patients with Hematologic Malignancies after Sars-Cov-2 Infection

Introduction:The spread of SARS-CoV-2 virus continues to pose a major public health threat. Patients with cancer are thought to be at increased risk from SARS-COV-2 infection due to the immunodeficiency that results from the underlying neoplasm and treatment. The immune response to this infection has been the subject of great interest, with an extreme variation in clinical severity between infected individuals. Variation in the immune cell response (B, T, and NK lymphocytes, monocytes, and myeloid derived suppressor cells (MDSCs), among others) and their function have been hypothesized to be responsible for this range of presentation.Methods:Two patients with a history of hematologic malignancies were matched with three non-cancer patients with similar baseline clinical characteristics and severity of COVID related illness. The critical group (CG) was defined as those requiring mechanical ventilation (MV) due to COVID related respiratory failure and the non-critical group (NCG) were hospitalized but did not require MV. All samples studied were obtained from peripheral blood and processed within 4-hours of collection. Peripheral blood mononuclear cell (PBMC) were isolated using ficoll density gradient separation. Flowcytometric analysis using CytekTM Aurora was done on fresh PBMC samples. Thirty antibody-based flow markers were used to identify 54 distinct immune cell populations. IRB approval was obtained.Results:Critical Group (CG):The CG included case 1, a 47 year-old (y.o.) female (F) with a history (hx) of acute myeloid leukemia and had an matched related donor allogeneic hematopoietic stem cell transplant (alloHSCT) 10-years prior remaining in remission, with hematologic recovery, and off immunosuppressants treated with remdesivir and coritcosteroids for COIVD directed therapy; and case 2, a 55 y.o. F with a hx of HIV treated with corticosteroids for COIVD directed therapy (see Figure 1a).Non-Critical Group (NCG):The NCG included case 3, a 73 y.o male (M) with hx of relapsed/refractory Philadelphia chromosome negative Acute Lymphoblastic Leukemia with loss of CD19 and CD22 expression following treatment with blinatumumab and inotuzumab, and most recently treated with decitabine/venetoclax; case 4, a 66 y.o. M with hx of cardiomyopathy; and case 6, a 54 y.o. M with hx of obesity. None of the NCG cases were treated with COVID directed therapy.See Table 1 for further clinical information.Immunophenotypic expression:Flow cytometry gating strategy done as outlined in Fig 1a. Case 1 had a high proportion of B-cells, CD8+ T-cells, and cells with exhaustion markers (CD8+CD94+ T-cells, CD4+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1-CD94+ NK T-cells, Lag3+Cd11b- non-TB leukocytes) and MDSC immunophenotypes compared with matched case 2. Case 3 also had a high proportion of exhaustion markers (Lag3+CD39 low B-cells, CD8+PD1+ T-cells, CD8+CD94+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1+CD94+ NK T-cells, PD1-CD94+ NK T-cells, Lag3+CD11b+, Lag3+CD11b- non-TB leukocytes) and high expression of immunosuppressive Treg and all MDSC; although high expression of granulocytic MDSC. Case 2 had a significant number of exhaustion and immunosuppressive cells as well. Cases 4 and 5 had a higher predominance of all T-cell subtypes and also had variable expression of exhaustion and immunosuppressive immunophenotypes (See Fib 1b).Conclusion:In our study of one critical and one non-critical patient with a history of hematologic malignancy matched with three non-cancer patients we demonstrate the high predominance of exhaustion markers (Lag3,PD1,CD94) and immunosuppressive cell types (Treg, granulocytic and monocytic MDSC). These findings are consistent with the fact that both CG and NCG, as hospitalized patients, represent the most severely ill COVID patient cohort. Of notable interest to the cancer population, cases 1 and 3 had a significant number of exhaustion and immunosuppressive immunophenotypes, suggestive of baseline exhaustion following alloHSCT even years after engraftment in case 1 and attenuated functional immunity in a patient undergoing active treatment in case 3. Interestingly, case 3 had lower expression of all MDSC, a known treatment effect of decitabine. Paired cytokine measurement and its effect on immunophenotype is underway. Additionally, we plan to present an atlas of the peripheral immune cell response on fifteen additional non-cancer COVID patients.DisclosuresNo relevant conflicts of interest to declare.

Cite-Seq Profiling of T Cells in Multiple Myeloma Patients Undergoing BCMA Targeting CAR-T or Bites Immunotherapy

Adaptive T cell therapy using chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEs) have demonstrated encouraging responses in heavily pre-treated multiple myeloma (MM) patients. However, the cellular and molecular predictors of clinical response are not fully understood as well as the mediators of acquired resistance remain elusive. Local immune suppression and T cell exhaustion are important mediators of responses therefore, it is plausible to speculate that a tolerant tumor microenvironment and the expansion of specific T cell populations may dictate clinical responses. In this study, we performed at the single cell level a broad immunophenotypic and transcriptomic characterization of the blood and bone marrow (BM) T cells of sensitive and resistant MM patients treated with adaptive T cell therapies. Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in order to identify cellular mediators of resistance to these adoptive immune therapies. Serial blood samples and BM aspirates (n=12) were collected from patients treated with anti-BCMA CAR-T or BCMA-CD3 BiTEs at variable time points, prior and post initiation of therapy and at relapse. Bone marrow mononuclear fractions were isolated through ficoll density gradients coupled with magnetic sorting of CD3pos T cells. Unbiased mRNA profiling coupled with feature barcoding technology for cell surface protein (TotalSeq-B) of BM CD3pos T cells was then performed by using the chromium single cell (10x Genomics). Paired-end sequencing was performed on Illumina platform. Cell Ranger and Seurat pipeline were used for sample de-multiplexing, barcode processing, single-cell 3′ gene counting, cell surface protein expression and data analysis. CAR-T cells were identified by the expression of the chimeric CAR-T cell transcript. The parallel measurement of transcripts and cell surface protein phenotypes of CD3pos T cells using a panel of 19 immune surface markers underlined the T cell repertoire diversity and identified different T cell subsets among the CD8pos and CD4pos T cells. Notably, the cell surface protein information overlaid on the transcript-generated UMA allowed accurate identification of all main immune clusters, in particular for the CD45RA and CD45RO positive cells. Comparison of CITE-Seq features revealed that the T cells composition of the blood and BM niches differed significantly between sensitive and resistant patients. As such an enrichment of CD4pos T cells with a higher CD4:CD8 ratio was noted in responding patients. Phenotypic (CD45RA, CD45RO, CD95, CCR7, CD62L, CD28, CD27) and transcriptional signatures (TCF7, LEF1, GATA3, EOMES, TBX21, PRDM1) also identified a higher proportion of memory like T cells (Tscm, Tcm) in responding patients. In contrast, T cells of resistant patients were enriched with terminally exhausted (Tex) and senescent cells with loss of CD28, high GMZHand GMZB, CD57pos, CD69pos and CD160pos as well as upregulation of TBX21. Expression of T cell checkpoint inhibitors such as LAG3, TIGIT and PD1 was high in these Tex cells as well as in some Tem. Of note, ex vivo T cell activation studies with TIGIT blockade demonstrated T cell activation in an autologous MM and T cell co-culture system with enhanced MM cells death. An expanded cluster of regulatory T cells (Treg) FOXP3pos,CD25pos was also observed in two resistant patients. Of note, no loss of BCMA transcript or surface expression was noted in MM cells at the time of acquired resistance. Single cell transcriptome of primary MM cells and chromatin accessibility (ATAC-seq) analyses of T cells of these patients are ongoing to investigate the transcriptional programs and epigenetic factors underlying the immune escape. Combined single cell features profiling of the transcriptome and surface protein expression of T cells from MM patients receiving BCMA targeted CAR-T or BiTEs therapies revealed potential mediators of resistance. In particular, T cells composition (low CD4:CD8 ratio and reduced population of Tscm, Tcm) along with an enrichment of terminally exhausted T cells are the main features observed in resistant patients. Delineating these mechanisms will guide future T cells engineering studies to enhance the efficacy and response durability of adoptive immunotherapy in MM. Disclosures McCulloch: Amgen: Honoraria; Sanofi: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Duggan:Jannsen: Consultancy; Amgen: Consultancy; Novartis: Honoraria; Celgene: Consultancy; Astra Zeneca: Consultancy. Jimenez-Zepeda:Janssen, Celgene, Amgen, Takeda: Honoraria. Bahlis:AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; BMS/Celgene and Janssen: Consultancy, Honoraria, Other: Travel, Accomodations, Research Funding; Karyopharm Therapeutics: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Neri:Celgene/BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

A Novel CD138-Targeting Monoclonal Antibody Induces Potent Myeloma Killing and Further Synergizes with IMiDs or Bortezomib in in Vitro and In Vivo Preclinical Models of Human Multiple Myeloma

Immunotherapeutically targeting CD138 has not been successfully translated into substantial clinical benefit in multiple myeloma (MM) patients. We here developed and determined in preclinical models of human MM the efficacy of VIS832, a novel humanized monoclonal antibody (MoAb) with differentiated CD138 target binding to the anti-CD138 MoAb BB4 within indatuximab ravtansine (BT082). Compared with BB4, VIS832 has significantly improved binding (P<.005, up to 1-log increases) to all MM cell lines (n>8). In the reporter-based antibody-dependent cellular cytotoxicity (ADCC) bioassay, EC50 of VIS832 was approximately 99.31 ng/ml (0.66 nM), whereas the BB4 possessing an isotype matched human IgG1 minimally induced ADCC against U266 MM cells. Evaluation of VIS832-induced ADCC in NK-MM cell co-cultures determined its EC50 values ranged from 2.22 + 0.37 to 15.3 + 2.71 ng/ml, with % maximal lysis of 37.06 + 1.45 % to 97.3 + 3.34 %, across tested MM cell lines (n=12), both sensitive or resistant to current therapies including dexamethasone, IMiDs, and bortezomib. While VIS832 effectively induced ADCC against parental cells with high and low CD138 levels, no lysis was detected in paired CD138-knockout MM cells. In the presence of BM stromal cells or osteoclasts, two key MM-supporting accessory cells in the BM milieu, VIS832-induced MM cytolysis was minimally affected. Importantly, VIS832 eliminated autologous MM cells of bone marrow (BM) samples from relapsed/refractory (RRMM) patients, with > 60% maximal lysis and > 90% killing in 4 out of 7 patient BM samples. EC50 values of VIS832 were 8.58-86.04 ng/ml in this patient cohort, comparable to those (17.28-179.3 ng/ml) of its non-humanized predecessor mAb 2810 against another RRMM patient cohort (n=6). In contrast to its potent autologous CD138+ patient cell lysis, VIS832 did not affect CD138-negative patient BM cells. VIS832 induced higher maximal lysis (~3-fold) of all target MM cell lines than CD38-targeting daratumumab (dara), regardless of resistance to lenalidomide and pomalidomide. These results also confirmed significantly higher CD138 vs CD38 levels in all MM cell lines and patient MM cells (P<.0001). Specifically, VIS832 remained active against MM cells resistant to dara, either via loss of CD38 or acquired drug resistance following extended dara treatment in exvivo co-cultures. Unlike dara, VIS832 selectively induced MM cell lysis via NK activation, without inducing NK cell apoptosis due to CD38 expression. When combined with lenalidomide, either pretreatment or added concomitantly, VIS832-induced MM cell lysis was significantly enhanced, with combination index (CI) of < 1 indicating synergism. Furthermore, antibody-dependent cellular phagocytosis (ADCP) represented another effector-mediated killing of VIS832 against MM cells, with phagocytosis dependent on both VIS832 and the E:T ratio. The ADCP activity of VIS832 was significantly higher than that observed for dara (P<.05) against MM cell lines sensitive or resistant to IMiDs and dara, and VIS832 induced significant ADCP against dara-resistant MM cells. Finally, in a disseminated MM1S xenograft model of human MM in CB-17 SCID mice, VIS832 alone reduced median tumor burden and improved overall survival from 30 to >60 days (P<.0001). This efficacy was sustained after discontinuation of treatment 3 weeks prior to study termination. Combined treatment of tumor-bearing mice with both VIS832 and bortezomib at sub-optimal doses rapidly eradicated tumors, resulting in 100% survival of all tumor-free animals to the end of study at day 73. The superior efficacy of combined VIS832 and bortezomib when compared to either agent alone suggests synergistic invivo activity, consistent with synergistically enhanced invitro cytotoxicity of this combination (CI < 1). Taken together, the significant invivo efficacy of VIS832, coupled with its mechanisms of action and potent in vitro MM cytotoxicity, strongly support clinical development of VIS832, as monotherapy and in combination, to overcome multidrug resistance and improve patient outcome in MM. Once its efficacy is established in RRMM, its favorable therapeutic index should allow for moving rapidly to earlier stages of disease, newly diagnosed MM and smoldering MM. Disclosures Chaganty: Visterra Inc: Current Employment. Ramakrishnan:Visterra Inc: Current Employment. Wollacott:Visterra Inc: Current Employment. Viswanathan:Visterra Inc: Current Employment. Adari:Visterra Inc: Current Employment. Munshi:Karyopharm: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Takeda: Consultancy; AbbVie: Consultancy. Babcock:Visterra Inc: Current Employment. Shriver:Visterra Inc: Current Employment. Myette:Visterra Inc: Current Employment. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

Peripheral Blood CD34+ Donor Chimerism Is Superior to CD3+ Donor Chimerism for Predicting Relapse Following Allogeneic Stem Cell Transplantation for Myeloid Malignancies

Background: Disease relapse remains the main cause of mortality after allogeneic stem cell transplantation (allo-SCT) for patients with myeloid malignancies. Loss of donor chimerism (DC) is commonly used as a biomarker of impending relapse, which allows the initiation of pre-emptive therapies such as withdrawal of immunosuppression or donor lymphocyte infusion (DLI). Surprisingly, there are few if any direct comparisons of peripheral blood CD34+ and CD3+ DC as biomarkers to predict relapse. We hypothesized that loss of CD34+ DC may be a more direct measure of impending relapse given most myeloid malignancies express CD34. Methods: We prospectively measured peripheral blood CD34+ and CD3+ DC on days 30, 60, 90, 120 and 180 following allo-SCT for patients with AML (n=113) or MDS (n=23) transplanted at a single centre between July 2011 and November 2019. Chimerism analysis was performed using purified cell subsets isolated from 60 mL peripheral blood using PCR-based amplification of short tandem repeats (STRs). The goal of this retrospective analysis was to compare the value of CD3+ and CD34+ DC for predicting relapse. Institutional practice for CD34+ DC below 80% included a bone marrow biopsy to identify morphologic relapse and donor lymphocyte infusion. Statistical analysis was performed with R 3.5.2 (The R project for Statistical computing) or GraphPad (v8.2.0). Results: Overall, 41 of 136 (30%) patients had morphologic relapse at a median time of 153 days after allo-SCT (range 50-1742). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of CD3+ and CD34+ donor chimerism for morphologic relapse is shown in Figure 1A. CD34+ DC outperformed CD3+ DC in all criteria, irrespective of the percentage chosen. Furthermore, a concurrent reduction in CD34+ DC was seen in almost all patients (14/16 at the 80% level) that had a fall in CD3+ DC. A fall in CD34+ DC below 80% was highly predictive for relapse, with a 5 year relapse free survival of only 17% compared with 80% for those that maintained DC > 80% for the first 180 days (Figure 1B). To determine the clinical utility of CD34+ DC, we measured the time to relapse in those patients that had a fall in DC without morphologic relapse at the time of DC measurement (Figure 1A). Based upon our institutional trigger of CD34+ DC < 80%, the median time to relapse was 49 days in 22 patients. In contrast, a CD34+ DC < 90% had a much longer time to relapse (71 days). However, this longer lead time would come at the price of unnecessary intervention in almost half of all patients as the PPV for CD34+ DC < 90% was only 55%. Loss of CD3+ DC had the longest lead time (> 70 days), however it was only useful up to 10 (24%) of all relapses. Finally, DLI administered for CD34+ DC < 80% maintained durable remission (> 12 months) in only 2 of 19 patients. Conclusion: This is the largest comparison of peripheral blood CD34+ and CD3+ DC following allo-SCT. Our results show that monthly monitoring of CD34+ DC in the first 6 months after allo-SCT is a more useful biomarker than CD3+ DC for predicting relapse in patients allografted for AML or MDS. The level of CD34+ DC chosen to trigger intervention should be guided by characteristics of the intervention such as toxicity and expected response time. Given the relative ineffectiveness of DLI for CD34+ < 80%, we suggest that 90% may provide greater time for immunologic responses. Figure Legend (A) Characteristics of different levels of CD3 and CD34 DC at any time in the first 180 days post-allo-SCT. Number of patients that fulfil the level in the total cohort of 136 patients. Number of patients before relapse indicates the number that do not have morphologic relapse at the time of DC measurement. Median days before relapse is shown for those patients. (B) Relapse free survival of patients according to CD34+ DC > 80% (blue line) or < 80% (yellow line) in the first 180 days. Disclosures Spencer: AbbVie, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Consultancy; Celgene, Janssen and Takeda: Speakers Bureau; Amgen, Celgene, Haemalogix, Janssen, Servier and Takeda: Research Funding; AbbVie, Amgen, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Honoraria. Wei:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; MacroGenics: Consultancy, Honoraria; Servier: Consultancy, Honoraria, Research Funding; Walter and Eliza Hall Institute: Other: former employee and receives a fraction of its royalty stream related to venetoclax; Pfizer: Honoraria; Genentech: Honoraria; Astra Zeneca: Honoraria, Research Funding; AbbVie Inc.: Consultancy, Honoraria, Research Funding.

Baseline Biomarkers of T-Cell Function Correlate with Clinical Responses to Odronextamab (REGN1979), and Loss of CD20 Target Antigen Expression Identified As a Mechanism of Treatment Resistance

Baseline Biomarkers of T-Cell Function Correlate with Clinical Responses to Odronextamab (REGN1979), and Loss of CD20 Target Antigen Expression Identified As a Mechanism of Treatment Resistance

Retreatment With Axicabtagene Ciloleucel (Axi-Cel) in Patients With Relapsed/Refractory Indolent Non-Hodgkin Lymphoma in ZUMA-5

Retreatment With Axicabtagene Ciloleucel (Axi-Cel) in Patients With Relapsed/Refractory Indolent Non-Hodgkin Lymphoma in ZUMA-5

Abstract IA08: Tracking the impact of tumor mutations in the T cell differentiation landscape of lung cancer

Abstract Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours and whether this affects patient outcomes is unknown. Here, we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets with strong phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states was associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC. Citation Format: Sergio A. Quezada. Tracking the impact of tumor mutations in the T cell differentiation landscape of lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA08.

The IMMENSE Study: The Interplay Between iMMune and ENdothelial Cells in Mediating Cardiovascular Risk in Systemic Lupus Erythematosus.

Patients with systemic lupus erythematosus (SLE) have a significant increase in cardiovascular (CV) risk although they display a preserved number of circulating angiogenic CD3+CD31+CXCR4+ T cells (Tang), a subpopulation of T cells which promotes repair of damaged endothelium. This happens due to the concomitant expansion of a Tang subset with immunosenescent features, such as the loss of CD28. Therefore, the aim of this study was to elucidate the interplay between Tang subpopulations and endothelial cells in a group of young SLE patients without previous cardiovascular events. Twenty SLE female patients and 10 healthy controls (HCs) were recruited. Flow cytometric analysis of endothelial progenitor cells (EPCs) and Tang subsets were performed and serum levels of interleukin (IL)-6, -8, matrix metalloproteinase (MMP)-9 and interferon (IFN)-γ were measured. Human umbilical vein endothelial cells (HUVECs) proliferation and pro-inflammatory phenotype in response to subjects’ serum stimulation were also evaluated. Results showed that the percentage of Tang and EPC subsets was reduced in SLE patients compared with HCs, with a marked increase of senescent CD28null cells among Tang subset. SLE disease activity index-2000 (SLEDAI-2K) was inversed related to Tang cells percentage. Furthermore, IL-8 serum levels were directly correlated with the percentage of Tang and inversely related to the CD28null Tang subsets. We indirectly evaluated the role of the Tang subset on the endothelium upon stimulation with serum from subjects with a low percentage of Tang CD3+ cells in HUVECs. HUVECs displayed pro-inflammatory phenotype with up-regulation of mRNA for IL-6, intercellular adhesion molecule (ICAM)-1 and endothelial leukocyte adhesion molecule (ELAM)-1. Cell proliferation rate was directly related to IL-8 serum levels and EPC percentage. In highly selected young SLE patients without previous CV events, we found that the deterioration of Tang compartment is an early event in disease course, preceding the development of an overt cardiovascular disease and potentially mediated by SLE-specific mechanisms. The overcome of the CD28null subset exerts detrimental role over the Tang phenotype, where Tang could exert an anti-inflammatory effect on endothelial cells and might orchestrate via IL-8 the function of EPCs, ultimately modulating endothelial proliferation rate.

A Case of Enteropathy Associated T-Cell Lymphoma (EATL) with Aberrant Phenotype, CD8+, CD56+: From Diagnosis to Death

Abstract Introduction/Objective Enteropathy Associated T-Cell Lymphoma (EATL) is a rare and aggressive subtype of primary intestinal T cell lymphoma which occurs in patients with Celiac Disease (CD), most prevalent in the western world with an incidence rate of 0.22-1.9 cases per 100,000. The classic immophenotype of these neoplastic intestinal T cells show loss of CD8 and CD56. The adherence to gluten-free diet, markedly decreases the incidence of this complication. Methods The patient’s previous and current biopsies, autopsy and EMR were reviewed. The patient is 66-year female that was diagnosed with Celiac Disease in 2003 after duodenal biopsy showed features suggestive of CD with positive anti-endomysial IgA antibody serology. She presents currently with burning abdominal pain, with subsequent CT scan showing a mass in the small bowel with mid gut rotation and lesions in the lungs, liver and bladder. Endoscopy showed multiple lesions extending from the hypopharynx to the large bowel, which on biopsy showed CD3+ lymphocytes expanding the lamina propria and infiltrating the crypt epithelium. Given the patient’s clinical history a diagnosis of EATL was made. The patient passed 5 days after diagnosis due to small bowel perforation. Results The initial duodenal biopsy showed villous blunting and increased intraepithelial lymphocytes. The biopsies of the gastrointestinal lesions show abnormal infiltrate of pleomorphic, intermediate in size lymphocytes with round to irregular and occasionally cleaved nuclei with pale to clear cytoplasm. These cells infiltrate the crypt epithelium. The immophenotype of these neoplastic cells are positive for CD3, CD7, CD8, CD56, TIA-1 and BF1, while negative for CD4, CD5 and CD30. T cell clonality was also positive. In addition to the above lesions, autopsy revealed involvement of an area of small bowel perforation with full-thickness mucosal wall involvement by the neoplastic cells. In addition, there is widely dissemenated disease involving the lung, liver, bone marrow, spleen, mesenteric lymph nodes, omentum, bladder, ovaries and myometrium (first report of uterine involvement). Conclusion CD8 positive EATL may occur in 19-30% of cases, and increases up to 50% in refractory CD. The differential diagnosis of MEITL, which is typically CD8 positive, is important and most be distinguished on the basis of clinical setting in the presence of Celiac Disease.

Failure of CD4 T Cell-Deficient Hosts To Control Chronic NontyphoidalSalmonellaInfection Leads to Exacerbated Inflammation, Chronic Anemia, and Altered Myelopoiesis

Immunocompromised patients are more susceptible to recurrent nontyphoidal Salmonella (NTS) bacteremia. A key manifestation of HIV infection is the loss of CD4 T cells, which are crucial for immunity to Salmonella infection. We characterized the consequences of CD4 T cell depletion in mice where virulent Salmonella establish chronic infection, similar to chronic NTS disease in humans. Salmonella-infected, CD4-depleted 129X1/SvJ mice remained chronically colonized for at least 5 weeks, displaying increased splenomegaly and more severe splenitis than infected mice with CD4 T cells. Mature erythrocytes, immature erythroid cells, and phagocytes accounted for the largest increase in splenic cellularity. Anemia, which is associated with increased mortality in Salmonella-infected humans, was exacerbated by CD4 depletion in infected mice and was accompanied by increased splenic sequestration of erythrocytes and fewer erythropoietic elements in the bone marrow, despite significantly elevated levels of circulating erythropoietin. Splenic sequestration of red blood cells, the appearance of circulating poikilocytes, and elevated proinflammatory cytokines suggest inflammation-induced damage to erythrocytes contributes to anemia and splenic retention of damaged cells in infected animals. Depleting CD4 T cells led to increased myeloid cells in peripheral blood, spleen, and bone marrow, as well as expansion of CD8 T cells, which has been observed in CD4-depleted humans. This work describes a mouse model of Salmonella infection that recapitulates several aspects of human disease and will allow us to investigate the interplay of innate and adaptive immune functions with chronic inflammation, anemia, and susceptibility to Salmonella infection.

Steatohepatitis Impairs T-cell–Directed Immunotherapies Against Liver Tumors in Mice

Nonalcoholic steatohepatitis causes loss of hepatic CD4+ T cells and promotes tumor growth. The liver is the most common site of distant metastases from a variety of malignancies, many of which respond to immunotherapy. We investigated the effects of steatohepatitis on the efficacy of immunotherapeutic agents against liver tumors in mice. Steatohepatitis was induced by feeding C57BL/6NCrl or BALB/c AnNCr mice a methionine and choline-deficient diet or a choline-deficient l-amino acid-defined diet. Mice were given intrahepatic or subcutaneous injections of B16 melanoma and CT26 colon cancer cells, followed by intravenous injections of M30-RNA vaccine (M30) or intraperitoneal injections of an antibody against OX40 (aOX40) on days 3, 7, and 10 after injection of the tumor cells. We measured tumor growth and analyzed immune cells in tumor tissues by flow cytometry. Mice were given N-acetylcysteine to prevent loss of CD4+ T cells from liver. Administration of M30 and aOX40 inhibited growth of tumors from intrahepatic injections of B16 or CT26 cells in mice on regular diet. However, M30 and/or aOX40 did not slow growth of liver tumors from B16 or CT26 cells in mice with diet-induced steatohepatitis (methionine and choline-deficient diet or choline-deficient l-amino acid-defined diet). Steatohepatitis did not affect the ability of M30 to slow growth of subcutaneous B16 tumors. In mice with steatohepatitis given N-acetylcysteine, which prevents loss of CD4+ T cells, M30 and aOX40 were able slow growth of hepatic tumors. Flow cytometry analysis of liver tumors revealed reduced CD4+ T cells and effector memory cells in mice with vs without steatohepatitis. Steatohepatitis reduces the abilities of immunotherapeutic agents, such as M30 and aOX40, to inhibit tumor liver growth by reducing tumor infiltration by CD4+ T cells and effector memory cells. N-acetylcysteine restores T-cell numbers in tumors and increases the ability of M30 and aOX40 to slow tumor growth in mice.

The Thymus in Chagas Disease: Molecular Interactions Involved in Abnormal T-Cell Migration and Differentiation.

Chagas disease, caused by the protozoan parasite T. cruzi, is a prevalent parasitic disease in Latin America. Presently, it is spreading around the world by human migration, thus representing a new global health issue. Chronically infected individuals reveal a dissimilar disease progression: while nearly 60% remain without apparent disease for life, 30% develop life-threatening pathologies, such as chronic chagasic cardiomyopathy (CCC) or megaviscerae. Inflammation driven by parasite persistence seems to be involved in the pathophysiology of the disease. However, there is also evidence of the occurrence of autoimmune events, mainly caused by molecular mimicry and bystander activation. In experimental models of disease, is well-established that T. cruzi infects the thymus and causes locally profound structural and functional alterations. The hallmark is a massive loss of CD4+CD8+ double positive (DP) thymocytes, mainly triggered by increased levels of glucocorticoids, although other mechanisms seem to act simultaneously. Thymic epithelial cells (TEC) exhibited an increase in extracellular matrix deposition, which are related to thymocyte migratory alterations. Moreover, medullary TEC showed a decreased expression of AIRE and altered expression of microRNAs, which might be linked to a disrupted negative selection of the T-cell repertoire. Also, almost all stages of thymocyte development are altered, including an abnormal output of CD4−CD8− double negative (DN) and DP immature and mature cells, many of them carrying prohibited TCR-Vβ segments. Evidence has shown that DN and DP cells with an activated phenotype can be tracked in the blood of humans with chronic Chagas disease and also in the secondary lymphoid organs and heart of infected mice, raising new questions about the relevance of these populations in the pathogenesis of Chagas disease and their possible link with thymic alterations and an immunoendocrine imbalance. Here, we discuss diverse molecular mechanisms underlying thymic abnormalities occurring during T. cruzi infection and their link with CCC, which may contribute to the design of innovative strategies to control Chagas disease pathology.

ABCL-122: Retreatment (reTx) of Patients with Refractory Large B Cell Lymphoma with Axicabtagene Ciloleucel (Axi-Cel) in ZUMA-1

Context: In ZUMA-1, the objective response rate with axi-cel, an autologous anti-CD19 CAR T cell therapy was 83% (58% complete response [CR] rate; Locke, et al. Lancet Oncol. 2019). However, approximately half of the responders relapsed, and the viability of reTx is unknown. Objective: To assess outcomes of axi-cel reTx in ZUMA-1. Methods: Patients with progressive disease (PD) were eligible for reTx if there was no evidence of CD19 loss by local review and if they had no DLTs in Phase 1 or comparable toxicities in Phase 2. Patients received 2 × 106 CAR T cells/kg after conditioning chemotherapy. Results: Thirteen patients received axi-cel reTx. Before 1st Tx, 69% of patients had an IPI 3-4, 85% had disease stage 3-4, and patients had a median 3 prior regimens. At 1st Tx, 6 patients achieved CR, 6 had a partial response (PR), and 1 had stable disease (SD) before PD. Median duration of response (DOR) was 96 days (range, 56-274). There was no Grade ≥3 cytokine release syndrome (CRS; 6 patients each had Grade 1 and 2) or Grade 4-5 neurologic events (NEs; 2 patients had Grade 1, 1 had Grade 2, 7 had Grade 3). Upon reTx, 54% of patients achieved response (4 CR, 3 PR). Response to reTx was more common among patients with a CR at 1st Tx (83%; 4/6 CR, 1 PR, 1 SD) than in patients with a PR at 1st Tx (33%; 2/6 PR, 1 SD, 3 PD). Median DOR at reTx was 81 days (range, 1-225+). One patient remained in response 255 days post-reTx. Comparable CRS rates occurred with reTx as with 1st Tx. Fewer patients experienced NEs with reTx than 1st Tx, and those that occurred were of lower grade: 3 patients each had Grade 1 and 3, 1 patient had Grade 2. Peak CAR T cell expansion was lower upon reTx than 1st Tx (median, 4.3 vs 66.1 cells/μL blood). Conclusions: ReTx with axi-cel may have transient clinical efficacy in some patients, and CAR T cell expansion and severe NEs may be attenuated. Confirmatory studies are needed. Funding provided by Kite, a Gilead Company.

Characterization of KIR+ NKG2A+ Eomes- NK-like CD8+ Tcells and their decline with age in healthy individuals.

KIR+NKG2A + Eomes+ CD8+ T cells, which are preferentially found with a TEMRA (CD45RA + CCR7-) phenotype while having the capacity to rapidly produce IFN-γ in response to innate stimulation (IL-12 and IL-18), have been demonstrated to exist in human cord blood and the adult blood circulation. This highly responsive T-cell type was termed NK-like CD8+ T cells due to their capability to act in an innate immune fashion in mice similar to NK cells. However, KIR+NKG2A + CD8+ T cells that are Eomes- represent a small proportion of unconventional T cells that have not been described until now. We compare the distribution of the memory phenotypes and senescence-associated markers of two T-cell subsets by multicolor flow cytometry in 10 cord blood samples and 105 healthy individuals (HIs) ranging from 6 to 84 years of age. We found that the Eomes+ population has a higher differentiation degree than the Eomes- population. T cells in the Eomes- subset show proportionally less TEMRA phenotypes while instead preferentially displaying a more naïve and TCM phenotype. Furthermore, the Eomes- population was shown to linearly decrease with age, while the Eomes+ population exhibited more senescence-associated characteristics, such as CD57 expression and loss of CD28. Overall, the KIR+NKG2A + Eomes- CD8+ T-cell population shares similar characteristics with the Eomes+ population, although with a lower degree of differentiation, lower senescence marker expression, and a proportional decrease with age. Thus, we suspect that KIR+NKG2A + Eomes-CD8+ T cells may represent a less differentiated stage of the NK-like CD8+ T-cell subset.

Abstract 6714: Genetic screens identify T cell co-stimulation as a key modifier of the redirected cytotoxicity of bispecific T cell engager (BiTE®) molecules

Abstract BiTE® constructs redirect T cell cytotoxicity against cancer cells through simultaneous binding to CD3 on T cells and a tumor-associated antigen (TAA) on cancer cells. Blinatumomab, an anti-CD19 BiTE® molecule used to treat pre-B-ALL patients, leads to more than 40% complete remission rates. Only 30% of relapsed patients exhibit loss of CD19 on leukemia cells, indicating that the majority of patients acquire blinatumomab resistance independently of target loss. Understanding such mechanisms could lead to new predictive biomarkers and help improve current BiTE® immunotherapy. To gain insight into the mechanism of resistance to BiTE® cytotoxicity, we performed CRISPR screens to identify tumor-intrinsic genetic factors that modulate BiTE® molecule efficacy in vitro. Cas9 expressing human cancer cells transduced with a genome-wide sgRNA library were incubated with T cells and either a control BiTE® molecule that did not recognize the cancer cells, or an anti-TAA BiTE® molecule at a concentration that induced the lysis of ~90% of target cells. Next-gen sequencing was used to identify enriched or depleted sgRNA, indicative of genes whose loss confers resistance or susceptibility to BiTE®-mediated T cell killing. As expected, the most enriched candidates from the screen were involved in the expression, post-translational modification or surface presentation of the TAA targeted by the BiTE® molecules. In the case of a screen using an anti-MSLN BiTE® molecule, a panel of genes involved in GPI-anchor biosynthesis, required to anchor MSLN to the plasma membrane was uncovered. The screen also revealed genes known to promote immune evasion such as PD-L1 as well as novel genes whose roles in immunotherapy were not extensively studied before, including genes involved in apoptosis and the NF-κB pathway. One of the top enriched candidates was CD58, which encodes a co-stimulatory molecule that binds to CD2 on T cells. Genetic perturbation and pharmacological blockade of CD58-CD2 interaction significantly reduced BiTE®-mediated killing of target cells. Furthermore, re-establishment of CD58-CD2 interaction either by overexpressing CD58 in cancer cells or by using CD2-agonist large molecules improved T cell activation and target cell killing. Moreover, concurrent blocking of CD2 and CD28 co-stimulation inhibited BiTE®-mediated killing more profoundly than blocking either pathway alone, indicating redundancy between CD58- and CD80/86-mediated co-stimulation. Our study reveals an important role of T cell co-stimulatory signals in BiTE®-mediated cytotoxicity. Considering that loss of CD58 is highly prevalent in DLBCL and relapsed Hodgkin lymphoma patients, it could potentially serve as a biomarker to predict response to BiTE®-based therapies. Our study also demonstrates that restoring CD58-CD2 interaction could sensitize cancer cells to BiTE®-mediated killing. Citation Format: Ye Shen, Patrick Collins, Jason Eng, Flordeliza Fajardo, Wendy Zhong, Olivier Nolan-Stevaux. Genetic screens identify T cell co-stimulation as a key modifier of the redirected cytotoxicity of bispecific T cell engager (BiTE®) molecules [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6714.