The Ratio of Costimulatory Vs Coinhibitory Molecules on AML Cells Determines the CD33-BiTE® Mediated T-Cell Response

Abstract Bispecific T-cell engagers (BiTE® antibody constructs) represent a novel immunotherapeutic strategy relying on the recruitment of T cells against tumor cells independent of TCR specificity. In Acute Myeloid Leukemia (AML), CD33 represents a suitable target antigen with high expression levels in >90 % of primary AML samples (Krupka et al, 2014). A CD33-BiTE® antibody construct (AMG 330) was developed mediating cytotoxicity against primary AML in vitro although to a variable degree (Krupka et al, 2016). Several parameters have been identified which modulate AMG 330-mediated cytotoxicity, including CD33 expression level as well as effector to target cell (E:T) ratio. However, the exact mechanism of T-cell activation through BiTE® antibody constructs is only partly understood. Physiological T-cell activation is based on engagement of the T-cell receptor complex together with costimulatory molecules whereas the absence of positive costimulation leads to T-cell anergy. In line with this concept, we hypothesized that BiTE®-mediated cytotoxicity requires positive costimulatory signals on the target cells for T-cell activation. We hypothesize that the ratio of costimulatory and coinhibitory molecules on AML cells determines the susceptibility to AMG 330-mediated cytotoxicity independent of target antigen expression level. A stable expression system was established utilizing murine Ba/F3 cells expressing human CD33 ± CD80 ± CD86 ± PD-L1. Co-cultures of Ba/F3 constructs and T cells were performed in presence of AMG 330 or a control BiTE® (cBiTE®) (5 ng/ml). For some experiments, T cells were separated into naive (CD45RA+/CCR7+) vs memory (CD45RADIM) cells using fluorescence-activated cell sorting. After 3 days, specific lysis was determined by flow cytometry and calculated as % specific lysis = 100 × (1 - live CD33+ cellsAMG 330 / live CD33+ cellscBiTE). T-cell proliferation was defined as number of CD2+ cells on day 3 compared to day 0. The expression pattern of CD33, CD80, CD86 and PD-L1 on primary AML cells was evaluated by specific fluorescence intensity (SFI) using multiparameter flow cytometry. A sample was considered positive at an SFI of > 1.5. Characterized primary AML patient samples were used in a long-term culture assay to determine the influence of the checkpoint molecule expression profile on AMG 330-mediated cytotoxicity. CD33 single positive Ba/F3 cells were not lysed upon the addition of AMG 330 and allogeneic T cells. Cytotoxicity could be restored by expression of CD80, CD86 and CD80+CD86 with following tendency: CD80+CD86 >> CD80 > CD86 (see table 1). There was a direct correlation of T-cell proliferation to AMG 330 mediated cytotoxicity. Memory T cells showed increased cytotoxicity compared to naive T cells against the different Ba/F3 cell lines. The influence of co-inhibition was investigated by additionally transducing PD-L1 into the different Ba/F3 cells. This led to a reduced AMG 330-mediated cytotoxicity in all PD-L1 expressing Ba/F3 cells (Table 1). This was accompanied by a reduction in T-cell proliferation. Looking at the expression profile of CD80 and CD86 in primary AML samples, we observed expression of CD80 in 7/123 and of CD86 in 188/226 of cases (respectively 5.7 % and 83.2 %). When comparing AMG 330-mediated cytotoxicity against primary AML cells for patient pairs with similar CD33 expression levels, a higher CD86/PD-L1 ratio led to an increased AMG 330-mediated cytotoxicity compared to patient samples with a lower CD86/PD-L1 ratio (exemplary data: SFI CD33+: 81.7; SFI-ratio CD86/PD-L1: 4; specific cytotoxicity: 64.2 % vs. SFI CD33+: 89.5; SFI-ratio CD86/PD-L1: 15.9; specific cytotoxicity: 96.4 %). In summary, this data supports the hypothesis that AMG 330-mediated cytotoxicity and T-cell proliferation are influenced by the ratio of costimulatory and coinhibitory molecules on AML cells. Our data supports the notion that the checkpoint profile on AML, rather than one molecule by itself, determines T-cell response to AMG 330. Prospective analyses in clinical trials are needed to validate the relevance of checkpoint molecules on target cells as a predictive biomarker for response. Disclosures Marcinek: AMGEN Research Munich: Research Funding. Brauchle:AMGEN Inc.: Research Funding. Kischel:AMGEN: Employment. Kufer:AMGEN Research Munich: Employment. Subklewe:Pfizer: Membership on an entity's Board of Directors or advisory committees; Roche AG: Research Funding; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Costimulation by B7 Modulates Specificity of Cytotoxic T Lymphocytes: A Missing Link That Explains Some Bystander T Cell Activation

Journal of Experimental Medicine ◽

10.1084/jem.186.10.1787 ◽

1997 ◽

Vol 186 (10) ◽

pp. 1787-1791 ◽

Cited By ~ 10

Author(s):

Pan Zheng ◽

Yang Liu

Keyword(s):

Cell Proliferation ◽

T Cells ◽

Influenza Virus ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Proliferation ◽

Target Cells ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation

It has been proposed that some bystander T cell activation may in fact be due to T cell antigen receptor (TCR) cross-reactivity that is too low to be detected by the effector cytotoxic T lymphocyte (CTL). However, this hypothesis is not supported by direct evidence since no TCR ligand is known to induce T cell proliferation and differentiation without being recognized by the effector CTL. Here we report that transgenic T cells expressing a T cell receptor to influenza virus A/NT/68 nucleoprotein (NP) 366-374:Db complexes clonally expand and become effector CTLs in response to homologous peptides from either A/PR8/34 (H1N1), A/AA/60 (H2N2), or A/NT/68 (H3N2). However, the effector T cells induced by each of the three peptides kill target cells pulsed with NP peptides from the H3N2 and H2N2 viruses, but not from the H1N1 virus. Thus, NP366–374 from influenza virus H1N1 is the first TCR ligand that can induce T cell proliferation and differentiation without being recognized by CTLs. Since induction of T cell proliferation was mediated by antigen-presenting cells that express costimulatory molecules such as B7, we investigated if cytolysis of H1N1 NP peptide–pulsed targets can be restored by expressing B7-1 on the target cells. Our results revealed that this is the case. These data demonstrated that costimulatory molecule B7 modulates antigen specificity of CTLs, and provides a missing link that explains some of the bystander T cell activation.

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The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-130197 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3202-3202

Author(s):

Cameron S. Bader ◽

Henry Barreras ◽

Casey O. Lightbourn ◽

Sabrina N. Copsel ◽

Dietlinde Wolf ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Activation ◽

Cd8 T Cell ◽

Advisory Committees ◽

Hematopoietic Stem

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells, leading to damage of particular host tissues characteristic of GVHD. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to pro-inflammatory cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) aHSCT models. Here we show that STING rapidly promotes donor CD8+ T cell activation and recipient APC death early after aHSCT. To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of MUD C3H.SW bone marrow (BM) + T cells into irradiated B6 wildtype (WT) or STING-/- recipients. Colon tissue from STING-/- recipients had >2x reduction in IFNβ, TNFα and IL-6 mRNA vs WT. MUD STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 wks post-HSCT vs WT. Double chimerism studies showed that the absence of STING in non-hematopoietic cells was responsible for GVHD amelioration. Conversely, a single dose of the highly specific STING agonist DMXAA given in vivo increased IFNβ, TNFα and IL-6 mRNA expression in WT, but not STING-/-, colon tissue 48 hrs after transplant and increased GVHD scores and lethality post-HSCT. Post-transplant cytoxan treatment abolished the ability of DMXAA to augment GVHD, supporting the notion that STING signaling increases donor T cell activation during aHSCT. To evaluate the potential impact of STING in the clinical setting, we transplanted C3H.SW BM + T cells into mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STINGHAQ/HAQ) and found that these mice also exhibited diminished GVHD. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD aHSCT contrasts to reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) aHSCT (Fischer J, et al, Sci. Transl. Med. 2017). Since CD4+ and CD8+ T cells are central in MMUD and MUD GVHD, respectively, we hypothesized that STING's effect on the predominant T cell subset in each model may explain these seemingly paradoxical results in STING-/- vs WT recipients. Therefore, we transplanted MMUD BALB/c BM + CD8+ T cells into B6-WT and STING-/- mice and found that - in contrast to MMUD recipients of combined CD4+ and CD8+ T cells - STING-/- recipients developed lower GVHD clinical scores, reduced skin pathology and had lower frequencies of activated T cells 8 wks post-HSCT vs WT, supporting a role for STING in the promotion of CD8+ T cell-mediated GVHD. Next, we investigated if recipient APCs played a role in STING's enhancement of CD8+ T cell-mediatedGVHD. We found that STING-/- mice had greater frequencies and numbers of recipient splenic CD11b+CD11c+ APCs 1 day after MMUD B6 into BALB/c aHSCT (Fig. A). BALB/c-STING-/- APCs also expressed reduced MHC class I protein levels (Fig. B). Moreover, STING-/- recipient spleens contained lower numbers of donor CD8+ T cells producing IFNγ and TNFα (Fig. C). These data support the hypothesis that STING contributes to early activation of donor CD8+ T cells and elimination of recipient APCs. Next, to identify if the loss of host MHC II+ APCs affected subsequent donor CD4+ T cell activation, B6-Nur77GFP transgenic donor T cells were used to explicitly monitor T cell receptor signaling. Consistent with increased numbers of host MHC II+ APCs in the spleens of STING-/- recipients 1 day post-aHSCT, we found greater frequencies and numbers of donor Nur77GFP CD4+ T cells expressing GFP, CD69 and IFNγ in STING-/- spleens 6 days after transplant (Fig. D). In summary, our studies demonstrate that STING plays an important role in regulating aHSCT and provide one potential mechanism by which STING could promote CD8+ T cell-mediated GVHD yet diminish CD4+-mediated GVHD. Overall, our studies suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. Disclosures Blazar: BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding. Levy:Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

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Clinical Efficacy of Anti-CD22 Chimeric Antigen Receptor T Cells for B-Cell Acute Lymphoblastic Leukemia Is Correlated with the Length of the Scfv Linker and Can be Predicted Using Xenograft Models

Blood ◽

10.1182/blood.v130.suppl_1.807.807 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 807-807

Author(s):

Marco Ruella ◽

Shannon L Maude ◽

Boris Engels ◽

David M. Barrett ◽

Noelle Frey ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Research Funding ◽

Cell Activation ◽

Lymphoblastic Leukemia ◽

Advisory Committees ◽

Xenograft Models

Abstract Introduction. Anti-CD19 chimeric antigen receptor T cells (CART19 or CTL019) have shown impressive clinical activity in B-cell acute lymphoblastic leukemia (B-ALL) and are poised to receive FDA approval. However, some patients relapse after losing CD19 expression. Since CD22 remains highly expressed in relapsed/refractory (r/r) B-ALL even in these patients, anti-CD22 CART (CART22) have been developed. The National Cancer Institute (NCI) reported 4/9 complete remission (CR) in patients receiving CART22, with 100% CR at the highest T cell dose (NCT02315612)(S hah NN, ASH 2016 #650). Patients and Methods. We generated a second-generation CAR22 differing from that used by the NCI only by the use of a longer linker [4x(GGGGS); LL vs. 1x(GGGGS); SL] between the light and heavy chains of the scFv (Fig. 1 A). This construct was tested in two pilot clinical trials in adults (NCT02588456)and children with r/r-ALL (NCT02650414). CART22 cells were generated using lentiviral transduction as in our previous studies. The protocol-specified CART22 dose was 2x106-1x107 cells/kg for pediatric patients <50kg and 1-5x108 for pediatric patients ≥50kg and adult patients,. infused after lymphodepleting chemotherapy. Patient characteristics are described in Table 1. For the adult trial, 5 patients were screened, 4 enrolled (1 patient withdrew consent) and 3 infused (1 manufacturing failure). For the pediatric trial, 9 patients were screened, 8 enrolled (1 screen failure) and 6 infused (two patients were not infused for disease progression). For the preclinical studies, we generated CART22LL and CART22SL and tested them in vivo using xenograft models. NOD-SCID gamma chain deficient (NSG) mice were engrafted with either a luciferase+ standard B-ALL cell line (NALM6) or primary B-ALL cells obtained from a patient relapsing after CART19 (CHP110R). We also used 2-photon imaging to study the in vivo behavior and immune synapse formation and flow cytometry to asses T cell activation. Results. CART22 cells were successfully manufactured for 10/12 patients. In the adult cohort 3/3 patients developed CRS (gr.1-3) and no neurotoxicity was observed; in the pediatric cohort out of 5 evaluable patients (1 discontinued for lineage switch to AML on pre-infusion marrow), 3/5 developed cytokine-release syndrome (CRS) (all grade 2) and 1 patient had encephalopathy (gr.1). CART22 cells expanded in the PB with median peak of 1977 (18-40314) copies/ug DNA at day 11-18. Interestingly, in an adult patient who had previously received CART19 a second CART19 re-expansion was observed following CART22 expansion (Fig 1 B). At day 28, in the adult cohort the patient who was infused in morphologic CR remained in CR, while the other 2 had no response (NR); in the pediatric cohort 2/5 patients were in CR, 1 in partial remission (PR) that then converted to CR with incomplete recovery at 2 months, and 2 NR. No CD22-negative leukemia progression was observed. Since our results with a long linker appeared inferior compared to the previously reported CART22 trial (short linker), we performed a direct comparison of the 2 different CAR22 constructs. In xenograft models, CART22SL significantly outperformed CART22LL (Fi 1 C) with improved overall survival. Moreover, CART22SL showed higher in vivo proliferation at day 17 (Fig 1 D). Mechanistically, intravital 2-photon imaging showed that CART22SL established more protracted T cell:leukemia interactions than did CART22LL, suggesting the establishment of productive synapses (Fig 1 E). Moreover, in vivo at 24 hrs higher T cell activation (CD69, PD-1) was observed in CART22SL from the BM of NALM-6-bearing mice. Conclusions. Here we report the results of two pilot clinical trials evaluating the safety and feasibility of CART22 therapy for r/r B-ALL. Although feasible and with manageable toxicity CART22LL led to modest clinical responses. Preclinical evaluation allowed us to conclude that shortening the linker by 15 amino acids significantly increases the anti-leukemia activity of CART22, possibly by leading to more effective interactions between T cells and their targets. Finally, with the caveats of cross-trial comparison, our data suggest that xenograft models can predict the clinical efficacy of CART products and validate the use of in vivo models for lead candidate selection Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Maude: Novartis Pharmaceuticals: Consultancy, Other: Medical Advisory Boards. Engels: Novartis: Employment. Frey: Novartis: Research Funding. Lacey: Novartis: Research Funding; Genentech: Honoraria. Melenhorst: Novartis: Research Funding. Brogdon: Novartis: Employment. Young: Novartis: Research Funding. Porter: Incyte: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Immunovative Therapies: Other: Member DSMB; Genentech/Roche: Employment, Other: Family member employment, stock ownship - family member; Servier: Honoraria, Other: Travel reimbursement. June: WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Grupp: Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; University of Pennsylvania: Patents & Royalties; Adaptimmune: Consultancy. Gill: Novartis: Patents & Royalties, Research Funding.

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CD20-TCB, a Novel T-Cell-Engaging Bispecific Antibody, Induces T-Cell-Mediated Killing in Relapsed or Refractory Non-Hodgkin Lymphoma: Biomarker Results From a Phase I Dose-Escalation Trial

Blood ◽

10.1182/blood-2019-123979 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5319-5319 ◽

Cited By ~ 2

Author(s):

Ann-Marie E Bröske ◽

Ian James ◽

Anton Belousov ◽

Enrique Gomez ◽

Marta Canamero ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Board Of Directors ◽

T Cell Activation ◽

Research Funding ◽

Cell Activation ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Introduction: CD20-TCB (RG6026) is a novel T-cell-engaging bispecific (TCB) antibody with a '2:1' molecular format that comprises two fragment antigen binding regions that bind CD20 (on the surface of B cells) and one that binds CD3 (on the surface of T cells). CD20-TCB offers the potential for increased tumor antigen avidity, rapid T-cell activation, and enhanced tumor cell killing versus other bispecific formats. The safety, tolerability, pharmacokinetics, biomarkers, and antitumor activity of CD20-TCB are currently being investigated in a multicenter Phase I dose-escalation trial (NP30179; NCT03075696). We recently presented preliminary clinical data demonstrating promising clinical activity in relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) patients with indolent or aggressive disease (Dickinson et al. ICML 2019). Here, we present preliminary blood and tissue biomarker analyses to explore modes of action, support optimal biological dose selection, and identify potential outcome predictors. Methods: For biomarker analyses, we performed immune profiling of peripheral blood by flow cytometry, analyzed plasma cytokine levels by ELISA, and characterized baseline and on-treatment tumor biopsies by immunohistochemistry/immunofluorescence assays and RNA sequencing. Biomarker data were obtained from 122 patients dosed with 0.005-25mg CD20-TCB. Results: CD20-TCB infusion led to a rapid and transient reduction in T cells in the peripheral circulation (T-cell margination) in all patients. T-cell margination reached nadir 6 hours after the first CD20-TCB infusion, and showed a strong association with CD20-TCB dose and receptor occupancy (RO%; as determined by Djebli et al. ASH 2019). Interestingly, rebound of T cells 160 hours after the first CD20-TCB infusion was associated with response to treatment. Responding patients showed long-term T-cell activation after the first infusion of CD20-TCB at doses from 0.6mg and above. T-cell activation was demonstrated by 2-4-fold elevation of T-cell activation markers such as Ki67, HLA-DR, PD-1, ICOS, OX40, and 4-1BB, which was sustained up to Cycle 5 (105 days). Analysis of paired pre- and on-treatment tumor biopsies (n=6) obtained before and 2-3 weeks after the first dose of CD20-TCB showed evidence of T-cell-mediated tumor cell killing. Analysis of archival and pre-treatment tumor biopsies (n=80) revealed that clinical responses were achieved irrespective of the amount of tumor T-cell infiltration at baseline. In contrast, preliminary baseline bulk tumor RNA sequencing data (n=46) showed upregulation of gene signatures associated with cell proliferation/Myc and T-cell subsets (effector vs exhausted-like) in non-responding patients. Conclusions: In this study, we demonstrated the mode of action of CD20-TCB, a novel bispecific antibody with promising clinical activity in R/R NHL. We also demonstrated that biomarker data on T-cell activation can support dose finding in conjunction with pharmacokinetics. Additional analysis is ongoing to evaluate response predictors and better characterize the population that will benefit most from T-cell mediated therapies. Disclosures Bröske: Roche: Employment, Equity Ownership. James:A4P Consulting Ltd: Consultancy. Belousov:Roche: Employment. Gomez:F. Hoffmann-La Roche Ltd: Employment. Canamero:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Ooi:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Grabole:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Wilson:F. Hoffmann-La Roche Ltd: Employment. Korfi:F. Hoffmann-La Roche Ltd: Consultancy. Kratochwil:F. Hoffmann-La Roche Ltd: Employment. Morcos:Roche: Employment, Equity Ownership. Ferlini:Roche: Employment, Equity Ownership. Thomas:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Dimier:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Moore:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Bacac:Roche: Employment, Equity Ownership, Patents & Royalties: Patents, including the one on CD20-TCB. Weisser:Pharma Research and Early Development Roche Innovation Center Munich: Employment, Equity Ownership, Patents & Royalties. Dickinson:Merck Sharpe and Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: CD20-TCB (also known as RG6026, RO7082859) is a full-length, fully humanized, immunoglobulin G1 (IgG1), T-cell-engaging bispecific antibody with two fragment antigen binding (Fab) regions that bind to CD20 (on the surface of B cells) and one that binds to CD3 (on the surface of T cells) (2:1 format). The 2:1 molecular format of CD20-TCB, which incorporates bivalent binding to CD20 on B cells and monovalent binding to CD3 on T cells, redirects endogenous non-specific T cells to engage and eliminate malignant B cells. CD20-TCB is an investigational agent.

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Galectin-3 Signaling in Donor T Cells Regulates Acute Graft Versus Host Disease (aGvHD) after Allogenic Transplantation

Blood ◽

10.1182/blood-2021-150777 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2765-2765

Author(s):

Hemn Mohammadpour ◽

Takemasa Tsuji ◽

Cameron R. MacDonald ◽

Joseph L. Sarow ◽

Jingxin Qiu ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd4 T Cells ◽

Research Funding ◽

T Cell Proliferation ◽

Advisory Committees ◽

Human T Cells ◽

Donor T Cells

Abstract Galectin-3 (Gal-3) is a unique member of the galectin family of lectins. Gal-3 possesses immune-regulatory functions depending on the immune cell and the immunologic situation. There are no studies that specifically delineate the role of Gal-3 in the setting of acute GvHD but mounting research suggests that dysregulation of pathways involving the galectin family may contribute to the pathogenesis of other immune disorders. Gal-3 is expressed by many types of immune cells, including T-cells. It suppresses signaling downstream of the TCR, decreases effector T-cell cytokine production, but increases the development and differentiation of memory T cells, myeloid cells, and macrophages. We investigated the mechanisms and downstream events of Gal-3 signaling in donor T cells after Allo-HCT, using Gal-3 knockout (Gal-3 -/-) mice. We further studied the effect of Gal-3 in controlling aGvHD incidence and severity while preserving the Graft-versus Leukemia (GvL) effect by overexpressing Gal-3 in human T cells. We utilized both a major MHC-mismatch (C57B/6 (H-2 b) into BALB/c (H-2 k) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2 b) into C3H/SW (H-2 b) model. Lethally irradiated recipient BALB/c and C3H/SW WT animals were injected with T cell depleted bone marrow alone (3 ×10 6) or with splenic T cells derived from allogeneic WT or Gal-3 -/- B6 donors (0.7 × 10 6 T cells in B6 → BALB/c and 1.5 × 10 6 in B6 → C3H/SW). We found that donor T cells express Gal-3 after Allo-HCT and that Gal-3 expression in WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, decreased clinical GvHD scores, and longer survival when compared to mice receiving Gal-3 -/- donor T cells (Figure 1A). We studied the mechanisms by which Gal-3 signaling controls the severity of aGvHD. Using flow cytometry analysis, we determined that Gal-3 plays a critical role in T cell proliferation and exhaustion. Gal-3 -/- T cells have a cytotoxic T phenotype with increased IFN-ℽ and GM-CSF production in T cells from the spleen and liver tissues on days 7 and 14 after Allo-HCT when compared to WT T cells (Figure 1B). There was a significant increase in T cell proliferation in Gal-3 -/- CD4 +T cells with a significantly higher level of IFN- ℽ mediated activation induced cell death (AICD) when compared to WT T cells. Gal-3 expression in T cells significantly increased the expression of exhaustion markers evidenced by a higher percentage of Slamf6 + Tim-3 + in WT T cells when compared to Gal-3 -/- T cells (Figure 1B). Gal-3 induced T cell exhaustion by through overactivation of NFAT signaling (data not shown). We sought to determine whether overexpression of Gal-3 in human T cells could control GvHD without affecting GVL. Gal-3 was overexpressed in human T cells using retrovirus containing Gal-3, vector alone and control T cells: Gal-3 T cells (T RV-Gal-3), GFP T cells (T RV-GFP) and control T cells were injected in irradiated NSG-HLA-A2 mice. All human cells expressed HLA-A2. Gal-3 overexpression in T cells effectively controlled the severity and mortality of GvHD after Allo-HCT in this humanized murine model of GvHD, evidenced by decreased body weight loss and decreased GvHD clinical scores in recipients transplanted with Gal-3 T cells when compared to control or GFP T cells (Figure 1C). Gal-3 overexpression did not impair the GvL effect when T cells cultured with Raji and THP-1 cell lines in vitro (data not shown). Gal-3 overexpression in T cells increased the frequencies of exhausted CD4 + T cells, and central memory CD4 + T cells while decreasing the percentage of effector CD4 T cell and INF-ℽ + CD4 + T cells. Clinical GI colon biopsies from patients undergoing allo-HCT were evaluated for Gal-3 expression in T cells using the multi-color Vectra 3 Automated Quantitative Pathology Imaging System. T cells in the colon biopsies expressed Gal-3. There was a significant correlation between Gal-3 MFI in CD4+ T cells, and GI histopathology score when analyzing Gal-3 intensity on Gal-3-expressing T cells. The Gal-3 MFI in CD4+ T cells was significantly lower in biopsies with higher colon GI histopathology scores (III-IV) compared to with lower colon GI histopathology scores I-II. In conclusion, these data reveal how Gal-3 can influence donor T cell proliferation and function in preclinical aGvHD models and point to the feasibility of manipulation of Gal-3 signaling to ameliorate aGvHD in the clinical setting. Figure 1 Figure 1. Disclosures Blazar: Rheos Medicines: Research Funding; Carisma Therapeutics, Inc: Research Funding; Equilibre Pharmaceuticals Corp: Research Funding; Tmunity Therapeutics: Other: Co-founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. McCarthy: Magenta Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Juno: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Evaluation of T Cell and Monocyte Immune Subsets in the Stem Cell Product of Patients Developing Engraftment Syndrome Following Autologous Stem Cell Transplantation for Multiple Myeloma and Lymphoma

Blood ◽

10.1182/blood-2018-99-120103 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4604-4604

Author(s):

Dante B. Descalzi-Montoya ◽

Zheng Yang ◽

Kira Goldgirsh ◽

Rena Feinman ◽

David S Siegel ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Activation ◽

Advisory Committees ◽

Classical Monocytes

Abstract BACKGROUND: High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is now standard of care for newly diagnosed patients with multiple myeloma (MM) and is used for some forms of non-Hodgkin lymphoma, providing improved outcomes. ASCT has been associated with a high incidence of engraftment syndrome (ES), which clinically presents as skin rashes, diarrhea, non-infectious fevers, and capillary leak syndrome in the peri-engraftment period. ES can be a severe and potentially lethal complication in patients who do not respond to corticosteroid therapy. RATIONALE: Prior to stem cell collection, MM and lymphoma patients are typically exposed to chemotherapy and immunomodulatory agents in order to reduce the cancer cell burden and other drugs to mobilize stem cells. It has been hypothesized that these agents may be involved with ES development in that they can disrupt the balance of immune regulatory and effector cell subsets. OBJECTIVE: To analyze T cell activation/memory and Treg cell compartments as well as classical, intermediate, and non-classical monocytes in the autologous apheresis product of MM and lymphoma patients undergoing ASCT. STUDY DESIGN: Samples were collected under IRB approval from 28 patients undergoing ASCT for the treatment of MM (n=21) or lymphoma (n=7) at the time of peripheral blood stem cell collection (the apheresis product); Peripheral blood mononuclear cells (PBMC) were isolated by sucrose gradient centrifugation and frozen until needed for phenotyping by multi-parametric flow cytometry. MM patients were conditioned for ASCT with melphalan alone (n=14) or in combination with bortezomib (n=7); lymphoma patients received conditioning with BEAM (carmustine, etoposide, cytarabine, and melphalan) chemotherapy. RESULTS: Of the 28 patients undergoing ASCT, 10 (35.7%) developed ES within the first four weeks post-transplantation. We analyzed by flow cytometric phenotyping the following immune cell subsets: overall CD4/CD8 T cells, CD4/CD8 naïve and memory cells, HLA-DR expression on memory T Cells, memory CD4 Tregs, as well as overall classical, intermediate, and non-classical monocytes. The absolute numbers (PBMC conc. (cell/ml of blood) x (% of cells in PBMC/100) and percentage population (# of events in population gate/# of total PBMC events x 100) data were transformed with log and square root functions, respectively. All data were tested for normality with a Shapiro-Wilks online test and p-values were obtained by performing an unpaired Student T-test. From our analysis of the apheresis product, the main cell compartments that were significantly increased in the ES+ group were the % CD8+ T cells [5.41 mean ± 0.51 s.e. vs. 3.34 ± 0.36 (ES-), p=0.003] and naïve CD8+ T cells [3.76 ± 0.56 vs. 1.61 ± 0.18 (ES-), p<0.001; In addition, although the memory CD8 T cell subset was not significantly increased in the apheresis product, the % of those memory CD8 T cells expressing HLA-DR significantly increased [2.0 ± 0.22 (ES+) vs. 1.2 ± 0.15 (ES-); p<0.01], suggesting an increase in CD8 T cell activation in the ES+ group. No major differences were observed in the CD4 memory Treg compartment or CD25 expression on CD4 T cells. In the monocyte compartment, the main subset that was significantly increased was the non-classical CD16+CD14low cells in the ES+ group [0.40 ± 0.08 vs. 0.25 ± 0.03 (ES-), p=0.02]. In addition, the % of CD25+ and CD163+ non-classical and intermediate monocytes were favorably increased in the ES+ group (non-classical monocytes, CD25+ [0.14 ± 0.03 vs. 0.07 ± 0.02 (ES-), p=0.03] and CD163+ [0.33 ± 0.06 vs. 0.18 ± 0.02 (ES-), p=0.03]; and for intermediate monocytes, CD25+ [0.44 ± 0.1 vs. 0.22 ± 0.04 (ES-), p=0.02] and CD163+ (p=0.02). No major differences were observed between ES+ and ES- groups for CD64 and PDL-1 expression. CONCLUSIONS: The development of ES correlated with the observation of a significant increase of naïve and activated CD8 T cells in the autologous apheresis product. In contrast, no significant differences were found between the ES+ and ES- groups in the CD4 T cell or memory Treg subsets, suggesting that they do not contribute to the etiology of the syndrome. Moreover, an increased presence of non-classical monocytes with higher expression of both CD163 and CD25 was found, suggesting increased potential for both M2 and M1 activity. Further investigation is needed to determine the implications of these findings for the development of ES. Disclosures Siegel: Merck: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Novartis: Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Biran:BMS: Research Funding; Merck: Research Funding; Amgen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau. Feldman:Johnson and Johnson: Speakers Bureau; KITE: Speakers Bureau; Seattle Genetics: Research Funding, Speakers Bureau; Pharmacyclics: Speakers Bureau; Janssen: Speakers Bureau; Portola: Research Funding; Celgene: Speakers Bureau. Skarbnik:Gilead Sciences: Honoraria, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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Anti-CD6 Monoclonal Antibody Itolizumab Efficiently Inhibits T Cell Proliferation after in Vitro TCR Stimulation in the Setting of Acute Graft Versus Host Disease

Blood ◽

10.1182/blood-2019-125721 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4517-4517 ◽

Cited By ~ 1

Author(s):

Benedetta Rambaldi ◽

Carol Reynolds ◽

Sharmila Chamling Rai ◽

Takeru Asano ◽

Yohei Arihara ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Research Funding ◽

Cell Activation ◽

T Cell Proliferation

CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation and trafficking and is central to inflammation. Early studies by Soiffer et al. demonstrated that ex vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreased the incidence of acute graft versus host disease (aGVHD), highlighting the importance of CD6+ cells in GVHD pathogenesis. Itolizumab, a humanized anti-CD6 monoclonal antibody, has been shown to modulate T cell activation and proliferation. The aim of this study was to characterize: (1) expression of CD6 and ALCAM, and (2) activity of itolizumab on T cell responses in peripheral blood from HCT patients pre- and post-aGvHD. We analyzed immune reconstitution in 31 adult patients who underwent HLA matched donor HCT for hematological malignancies. Patients received peripheral blood stem cell grafts and GVHD prophylaxis with tacrolimus and methotrexate. Twelve of 31 patients developed aGVHD at a median of 58 days, range 27-208, after HCT and systemic treatment was started in 83% of these cases. aGVHD grade severity was 25%, 58.3% and 16.7% of grade I, II and IV, respectively. Patient samples were collected at 1, 2 and 3 months after HCT and analyzed using multi-color flow cytometry. Nine healthy donors (HD) were analyzed as controls. Suppressive activity of itolizumab was tested using peripheral blood mononuclear cells (PBMC) obtained from HD and patients before (preGVHD) and after (postGVHD) aGvHD onset (within 30 days). PBMC were stimulated with antiCD3/CD2/CD28 coated beads in the presence of itolizumab or isotype control (cetuximab) for 72 hours. T cell proliferation was measured by CFSE dilution, while T cell activation and maturation was measured by expression of CD25 and CD45RO, respectively. For statistical analysis, non-parametric unpaired (Mann-Whitney) or paired (Wilcoxon matched-pairs signed rank) test were used. CD6+ T cells reconstituted early after transplant, accounting for 95% of positive CD3 T cells, range 57-100 at 1 month. Similar to HD PBMC, in the first 3 months after HCT, CD4 Tcon had the highest CD6 expression, while CD4 Treg had a lower CD6 expression compared to both CD4 Tcon and CD8 T cells (Fig 1A and 1B). To characterize the expression of CD6 on different T cell subsets, we used a t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm and visualized the data using a viSNE map (Fig 1C). Within the Tcon compartment, there were no differences in expression of CD6 between HD and patients at all 3 time points. Within CD4 Treg and CD8 T cells, CD6 expression was reduced in naïve CD8 T cells and CM Treg after transplant compared to HD. In HD, ALCAM expression was detected in 35% of CD14+ monocytes, 23% of CD19+ B cells, 20% of myeloid (CD11c+ CD123-) DCs and 97% of plasmacytoid (CD11c-CD123+) DCs. After HCT, expression of ALCAM in DC compartments was similar to HD. In functional studies, itolizumab inhibited CD4 and CD8 T cell proliferation in preGVHD samples, similar to HD controls. This effect was less prominent in samples collected from patients who had developed GVHD and were already receiving immunosuppressive medications, potentially confounding the ability to assess the effect of itolizumab in this assay (Fig 2A). Similar results were observed for CD25 (Fig 2B) and CD45RO (Fig 2C) expression pre- and post-aGVHD. Finally, itolizumab did not increase rates of cell death in samples from HCT patients as assessed by Annexin V expression, suggesting that itolizumab-mediated T cell inhibition was not due to increased T cell apoptosis. There was a slight increase in Annexin V expression in HD vs isotype control (21%, range 10-43 vs 15%, range 11-31, p= 0.0273). In conclusion, we demonstrate for the first time that CD6+ T cells reconstitute rapidly in peripheral blood after HCT and that CD6 expression is highest in Tcon while lowest in Treg (Tcon>CD8>Treg). Itolizumab efficiently inhibits T cell proliferation and activation after in vitro TCR stimulation of PBMC from aGvHD patients, thus representing a potential therapeutic for treating aGvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Rambaldi: Equillium: Research Funding. Koreth:Amgen: Consultancy; Cugene: Consultancy; Equillium: Consultancy. Cutler:Pharmacyclics: Consultancy; Omeros: Consultancy; Kadmon: Consultancy; BiolineRx: Other: DSMB; Cellect: Other: DSMB; Kalytera: Other: DSMB; ElsaLys: Consultancy; Genentech: Consultancy; BMS: Consultancy; Jazz: Consultancy; Incyte: Consultancy; Fate Therapeutics: Consultancy. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Ho:Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Soiffer:Jazz: Consultancy; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Mana therapeutic: Consultancy; Kiadis: Other: supervisory board. Ampudia:Equillium: Employment. Ng:Equillium: Employment, Equity Ownership. Connelly:Equillium: Employment, Equity Ownership. Ritz:Equillium: Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Aleta Biotherapeutics: Consultancy; Celgene: Consultancy; Avrobio: Consultancy; LifeVault Bio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy.

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Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit Anti-Thymocyte Globulin In Vitro

Frontiers in Immunology ◽

10.3389/fimmu.2020.592553 ◽

2020 ◽

Vol 11 ◽

Author(s):

Christian Binder ◽

Felix Sellberg ◽

Filip Cvetkovski ◽

Erik Berglund ◽

David Berglund

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Mixed Lymphocyte Reaction ◽

T Cell Proliferation ◽

Allogeneic Mixed Lymphocyte Reaction ◽

Dependent Cell

Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemtuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed via flow cytometry. Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.

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Activation Outcomes Induced in Naïve CD8 T-Cells by Macrophages Primed via “Phagocytic” and Nonphagocytic Pathways

Molecular Biology of the Cell ◽

10.1091/mbc.e07-07-0650 ◽

2008 ◽

Vol 19 (2) ◽

pp. 701-710 ◽

Cited By ~ 10

Author(s):

Isabel María Olazabal ◽

Noa Beatriz Martín-Cofreces ◽

María Mittelbrunn ◽

Gloria Martínez del Hoyo ◽

Balbino Alarcón ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Cell Activation ◽

T Cell Proliferation ◽

Microtubule Organizing Center ◽

Antigen Uptake ◽

Soluble Peptide

The array of phagocytic receptors expressed by macrophages make them very efficient at pathogen clearance, and the phagocytic process links innate with adaptive immunity. Primary macrophages modulate antigen cross-presentation and T-cell activation. We assessed ex vivo the putative role of different phagocytic receptors in immune synapse formation with CD8 naïve T-cells from OT-I transgenic mice and compared this with the administration of antigen as a soluble peptide. Macrophages that have phagocytosed antigen induce T-cell microtubule-organizing center and F-actin cytoskeleton relocalization to the contact site, as well as the recruitment of proximal T-cell receptor signals such as activated Vav1 and PKCθ. At the same doses of loaded antigen (1 μM), “phagocytic” macrophages were more efficient than peptide-antigen–loaded macrophages at forming productive immune synapses with T-cells, as indicated by active T-cell TCR/CD3 conformation, LAT phosphorylation, IL-2 production, and T-cell proliferation. Similar T-cell proliferation efficiency was obtained when low doses of soluble peptide (3–30 nM) were loaded on macrophages. These results suggest that the pathway used for antigen uptake may modulate the antigen density presented on MHC-I, resulting in different signals induced in naïve CD8 T-cells, leading either to CD8 T-cell activation or anergy.

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Human T cell activation. II. A new activation pathway used by a major T cell population via a disulfide-bonded dimer of a 44 kilodalton polypeptide (9.3 antigen).

Journal of Experimental Medicine ◽

10.1084/jem.161.6.1513 ◽

1985 ◽

Vol 161 (6) ◽

pp. 1513-1524 ◽

Cited By ~ 183

Author(s):

T Hara ◽

S M Fu ◽

J A Hansen

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Interleukin 2 ◽

Cell Subset ◽

Receptor Expression ◽

T Cell Proliferation ◽

Activation Pathway

In previous studies (17-21), monoclonal antibody (mAb) 9.3 has been shown to react with a major population of human T cells, which include T4+ helper/inducer T cells and T8+ cytotoxic T cells. In this investigation, mAb 9.3 was shown to precipitate a disulfide-bonded dimer of a 44 kD polypeptide. Comodulation experiments showed that this molecule is not linked to T3/Ti or T11 antigens. mAb 9.3 was capable of inducing T cell proliferation in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA). This effect was monocyte-independent. T cell activation with mAb 9.3 and TPA was associated with increases in interleukin 2(IL-2) receptor expression and IL-2 secretion. mAb 9.3 did not activate T cells, even with the addition of IL-1 or IL-2. Modulation of the T3 complex did not abolish mAb 9.3-induced T cell proliferation in the presence of TPA. These results suggest that the 9.3 antigen may serve as a receptor for an activation pathway restricted to a T cell subset.

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