Zinc Ionophore Pyrithione Mimics CD28 Costimulatory Signal in CD3 Activated T Cells.

Zinc is an essential trace element that plays a crucial role in T cell immunity. During T cell activation, zinc is not only structurally important, but zinc signals can also act as a second messenger. This research investigates zinc signals in T cell activation and their function in T helper cell 1 differentiation. For this purpose, peripheral blood mononuclear cells were activated via the T cell receptor-CD3 complex, and via CD28 as a costimulatory signal. Fast and long-term changes in intracellular zinc and calcium were monitored by flow cytometry. Further, interferon (IFN)-γ was analyzed to investigate the differentiation into T helper 1 cells. We show that fast zinc fluxes are induced via CD3. Also, the intracellular zinc concentration dramatically increases 72 h after anti-CD3 and anti-CD28 stimulation, which goes along with the high release of IFN-γ. Interestingly, we found that zinc signals can function as a costimulatory signal for T helper cell 1 differentiation when T cells are activated only via CD3. These results demonstrate the importance of zinc signaling alongside calcium signaling in T cell differentiation.

CD8+ T cells are necessary for improved sepsis survival induced by CD28 agonism in immunologically experienced mice.

Introduction: A hallmark of T cell dysregulation during sepsis is the downregulation of costimulatory molecules. CD28 is one of T cell costimulatory molecules significantly altered on memory T cells during sepsis. We recently showed that treatment with a αCD28 agonist in septic immunologically experienced mice led to improved survival. Therefore, here we aimed to identify the cell subset(s) necessary for the survival benefit observed in the context of CD28 agonism, and to further investigate the mechanism by which CD28 agonism improves sepsis survival in immunologically experienced mice. Methods: Mice received specific pathogen inoculation to generate memory T cell populations similar in frequency to that of adult humans. Once these infections were cleared and the T cell response had transitioned to the memory phase, animals were rendered septic via cecal ligation and puncture in the presence or absence of an agonistic anti-CD28 mAb. Results: Results demonstrated that CD8+ T cells, and not bulk CD4+ T cells or CD25+ regulatory T cells, were necessary for the survival benefit observed in CD28 agonist-treated septic immunologically experienced mice. Upon examination of these CD8+ T cells, we found that CD28 agonism in septic immunologically experienced mice was associated with an increase in Foxp3+ CD8+ T cells as compared to vehicle-treated controls. When CD8+ T cells were depleted in septic immunologically experienced mice in the setting of CD28 agonism, a significant increase in levels of inflammatory cytokines in the blood was observed. Discussion: Taken together, these results indicate that CD28 agonism in immunologically experienced mice effectively suppresses inflammation via a CD8+-dependent mechanism to decrease mortality during sepsis.

CD19-CD28: an affinity-optimized CD28 agonist for combination with glofitamab (CD20-TCB) as off-the-shelf immunotherapy.

ABSTRACT: Effective T-cell responses not only require the engagement of T-cell receptors (TCRs; "signal 1"), but also the availability of costimulatory signals ("signal 2"). T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513.

CD28 Superagonist D665-mediated activation of mouse regulatory T cells maintains their phenotype without loss of suppressive quality.

Regulatory T cells (Tregs) maintain immune homeostasis by regulating the activation of other immune cells. Preclinical studies show that the infusion of Tregs can promote immunological tolerance to allografts and prevent or cure multiple autoimmune diseases. However, Treg therapy is limited by high numbers of cells required to induce tolerance. In this study, we aimed at improving the in vitro expansion of sort purified mouse Tregs using the CD28 Superagonist (CD28-SA) D665 and comparing it to the conventional expansion using anti-CD3/anti-CD28 Dynabeads®. CD28-SA-stimulated Tregs expanded more than Dynabead®-stimulated Tregs while maintaining their phenotype by expressing the same level of CD4, CD25 and Foxp3. CD28-SA-expanded Tregs produced comparable amounts of IL-10 and TGFß while showing a slightly superior suppressive capacity compared to Dynabead®-stimulated Tregs. Thus, stimulating murine Tregs with the CD28-SA is a promising alternative since it maintains their suppressive capacity without altering their phenotype and yields a higher fold expansion within 14 days.

Immune reconstitution and clinical recovery following anti-CD28 antibody (TGN1412)-induced cytokine storm.

Cytokine storm can result from cancer immunotherapy or certain infections, including COVID-19. Though short-term immune-related adverse events are routinely described, longer-term immune consequences and sequential immune monitoring are not as well defined. In 2006, six healthy volunteers received TGN1412, a CD28 superagonist antibody, in a first-in-man clinical trial and suffered from cytokine storm. After the initial cytokine release, antibody effect-specific immune monitoring started on Day + 10 and consisted mainly of evaluation of dendritic cell and T-cell subsets and 15 serum cytokines at 21 time-points over 2 years. All patients developed problems with concentration and memory; three patients were diagnosed with mild-to-moderate depression. Mild neutropenia and autoantibody production was observed intermittently. One patient suffered from peripheral dry gangrene, required amputations, and had persistent Raynaud's phenomenon. Gastrointestinal irritability was noted in three patients and coincided with elevated γδT-cells. One had pruritus associated with elevated IgE levels, also found in three other asymptomatic patients. Dendritic cells, initially undetectable, rose to normal within a month. Naïve CD8+ T-cells were maintained at high levels, whereas naïve CD4+ and memory CD4+ and CD8+ T-cells started high but declined over 2 years. T-regulatory cells cycled circannually and were normal in number. Cytokine dysregulation was especially noted in one patient with systemic symptoms. Over a 2-year follow-up, cognitive deficits were observed in all patients following TGN1412 infusion. Some also had signs or symptoms of psychological, mucosal or immune dysregulation. These observations may discern immunopathology, treatment targets, and long-term monitoring strategies for other patients undergoing immunotherapy or with cytokine storm.

Predominant contribution of DGKζ over DGKα in the control of PKC/PDK-1-regulated functions in T cells.

Diacylglycerol kinase (DGK)-mediated consumption of the diacylglycerol (DAG) generated in response to antigen recognition is an important mechanism to limit T-cell function. Targeting DGK activity presents new opportunities for therapeutic manipulation of the immune response, but assessment of individual DGK functions is complex. T cells express two DGK isoforms, DGKα and DGKζ, and there are no isoform-specific inhibitors. Here we used short interfering RNA-mediated gene silencing in human T cells and DGKα- and DGKζ-deficient mice to define DGK isoform-specific regulation of key signaling pathways during T-cell activation. Our results identify DGKζ as the predominant brake on basal/tonic conditions as well as on downstream T-cell receptor/co-stimulatory signals. DGKζ silencing triggers basal RasGTP activation and facilitates enhanced membrane stability of protein kinase C alpha as well as increased activity of AGC kinases. Downstream of T-cell receptor/co-stimulation, DGKζ silencing results in enhanced and maintained recruitment of PKC theta to the membrane, as well as phosphoinositide-dependent protein kinase-1 activation and scaffolding functions. Our studies identify a previously unrecognized DGKζ contribution as a negative regulator of the crosstalk between phospholipase C-gamma- and phosphoinositide 3-kinase-regulated pathways. This DGKζ input helps to explain previous observations in DGK-deficient mice and suggests that the development of isoform-specific DGK inhibitors is of great interest for the manipulation of distinct aspects of T-cell responses.

Argonaute 2 immunoprecipitation revealed large tumor suppressor kinase 1 as a novel proapoptotic target of miR-21 in T cells.

MicroRNA (miR)-21 is an important suppressor of T-cell apoptosis that is also overexpressed in many types of cancers. The exact mechanisms underlying the antiapoptotic effects of miR-21 are not well understood. In this study, we used the Jurkat T-cell line as a model to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 reduced cell growth which could be explained by an increase in apoptosis. MicroRNA target gene identification by AGO2 RNA-immunoprecipitation followed by gene expression microarray (RIP-Chip) resulted in the identification of 72 predicted miR-21 target genes that were at least twofold enriched in the AGO2-IP fraction of miR-21 overexpressing cells. Of these, 71 were at least twofold more enriched in the AGO2-IP fraction of miR-21 overexpressing cells as compared to AGO2-IP fraction of control cells. The target gene for which the AGO2-IP enrichment was most prominently increased upon miR-21 overexpression was the proapoptotic protein LATS1. Luciferase reporter assays and western blot analysis confirmed targeting of LATS1 by miR-21. qRT-PCR analysis in primary T cells showed an inverse expression pattern between LATS1 transcript levels and miR-21 upon T-cell stimulation. Finally, LATS1 knockdown partially rescued the miR-21 inhibition-induced impaired cell growth. Collectively, these data identify LATS1 as a miR-21 target important for the antiapoptotic function of miR-21 in T cells and likely also in many types of cancer.

The rise and fall of the CD28 superagonist TGN1412 and its return as TAB08: a personal account.

Two decades ago, we discovered 'superagonistic' monoclonal antibodies specific for the CD28 molecule which are able to polyclonally activate T cells, in particular regulatory T cells, and are therapeutically active in many rodent models of autoimmunity, inflammation, transplantation, and tissue repair. A phase I trial of the human CD28 superagonist TGN1412 failed in 2006 due to an unexpected cytokine release syndrome, but after it became clear that dose-reduction allows to preferentially address regulatory T cells also in humans, clinical development was resumed under the name TAB08. Here, I recount the story of CD28 superagonist development from a personal perspective with an emphasis on the dramatic events during and after the 2006 phase I trial, the reasons for the failure of preclinical research to warn of the impending cytokine storm, and on the research which allowed resumption of clinical development.

In vivo activation of Treg cells with a CD28 superagonist prevents and ameliorates chronic destructive arthritis in mice.

Although regulatory T (Treg) cells are necessary to prevent autoimmune diseases, including arthritis, whether Treg cells can ameliorate established inflammatory disease is controversial. Using the glucose-6-phosphate isomerase (G6PI)-induced arthritis model in mice, we aimed to determine the therapeutic efficacy of increasing Treg cell number and function during chronic destructive arthritis. Chronic destructive arthritis was induced by transient depletion of Treg cells prior to immunization with G6PI. At different time points after disease induction, mice were treated with a CD28 superagonistic antibody (CD28SA). CD28SA treatment during the induction phase of arthritis ameliorated the acute signs of arthritis and completely prevented the development of chronic destructive arthritis. CD28SA treatment of mice with fully developed arthritis induced a significant reduction in clinical and histological signs of arthritis. When given during the chronic destructive phase of arthritis, 56 days after disease induction, CD28SA treatment resulted in a modest reduction of clinical signs of arthritis and a reduction in histopathological signs of joint inflammation. Our data show that increasing the number and activation of Treg cells by a CD28SA is therapeutically effective in experimental arthritis.

T-cell ligands modulate the cytolytic activity of the CD33/CD3 BiTE antibody construct, AMG 330.

Preclinical and emerging clinical studies demonstrate that bispecific T-cell engaging (BiTE) antibody constructs can potently lyse targeted tumor cells, but the determinants for their activity remain incompletely understood. Using human acute myeloid leukemia (AML) cell lines engineered to overexpress individual T-cell ligands, we found that expression of the inhibitory ligands, PD-L1 and PD-L2, reduced the cytolytic activity of the BiTE antibody construct targeting CD33, AMG 330; conversely, expression of the activating ligands, CD80 and CD86, augmented the cytotoxic activity of AMG 330. Consistent with these findings, treatment with an activating antibody directed at the co-stimulatory T-cell receptor, CD28, significantly increased AMG 330-induced cytotoxicity in human AML cell lines. Using specimens from 12 patients with newly diagnosed or relapsed/refractory AML, we found that activation of CD28 also increased the activity of AMG 330 in primary human AML cells (P=0.023). Together, our findings indicate that T-cell ligands and co-receptors modulate the anti-tumor activity of the CD33/CD3 BiTE antibody construct, AMG 330. These findings suggest that such ligands/co-receptors could serve as biomarkers of response and that co-treatment strategies with pharmacological modulators of T-cell receptor signaling could be utilized to further enhance the activity of this targeted therapeutic.

Role of acid sphingomyelinase bioactivity in human CD4+ T-cell activation and immune responses.

Acid sphingomyelinase (ASM), a lipid hydrolase enzyme, has the potential to modulate various cellular activation responses via the generation of ceramide and by interaction with cellular receptors. We have hypothesized that ASM modulates CD4(+) T-cell receptor activation and impacts immune responses. We first observed interactions of ASM with the intracellular domains of both CD3 and CD28. ASM further mediates T-cell proliferation after anti-CD3/CD28 antibody stimulation and alters CD4(+) T-cell activation signals by generating ceramide. We noted that various pharmacological inhibitors of ASM or knockdown of ASM using small hairpin RNA inhibit CD3/CD28-mediated CD4(+) T-cell proliferation and activation. Furthermore, such blockade of ASM bioactivity by biochemical inhibitors and/or molecular-targeted knockdown of ASM broadly abrogate T-helper cell responses. In conclusion, we detail immune, pivotal roles of ASM in adaptive immune T-cell responses, and propose that these pathways might provide novel targets for the therapy of autoimmune and inflammatory diseases.

In vitro polyclonal activation of conventional T cells with a CD28 superagonist protects mice from acute graft versus host disease.

Upon transplantation of T cells from a CD28 superagonist (CD28-SA) treated donor into an irradiated allogeneic host, the CD28-SA-induced activation and expansion of Treg cells inhibits acute graft versus host disease (aGvHD), while not abrogating the desired graft versus tumor effect. Human peripheral blood CD4(+) T cells, however, harbor only very few Treg cells. Therefore, we studied whether polyclonal in vitro prestimulation of conventional, that is Treg -cell-depleted, CD4(+) T cells of C57BL/6 mice with CD28-SA-coated paramagnetic beads is sufficient to protect recipient BALB/c mice from aGvHD. CD28-SA prestimulation of conventional CD4(+) T cells efficiently protected BALB/c recipient mice from aGvHD and CD28-SA-stimulated CD4(+) and CD8(+) T cells were capable of mediating long-term protection from the BCL1 lymphoma. The recently completed successful phase I testing of the human CD28-SA TGN1412/TAB08 should greatly facilitate further development of this straightforward method into a novel immunotherapy for patients.

CD28 superagonist-mediated boost of regulatory T cells increases thrombo-inflammation and ischemic neurodegeneration during the acute phase of experimental stroke.

While the detrimental role of non-regulatory T cells in ischemic stroke is meanwhile unequivocally recognized, there are controversies about the properties of regulatory T cells (Treg). The aim of this study was to elucidate the role of Treg by applying superagonistic anti-CD28 antibody expansion of Treg. Stroke outcome, thrombus formation, and brain-infiltrating cells were determined on day 1 after transient middle cerebral artery occlusion. Antibody-mediated expansion of Treg enhanced stroke size and worsened functional outcome. Mechanistically, Treg increased thrombus formation in the cerebral microvasculature. These findings confirm that Treg promote thrombo-inflammatory lesion growth during the acute stage of ischemic stroke.

Amplification of regulatory T cells using a CD28 superagonist reduces brain damage after ischemic stroke in mice.

BACKGROUND AND PURPOSE: Neuroinflammation plays an important role in ischemic brain injury. Regulatory T cells (Treg) are important endogenous immune modulators. We tested the hypothesis that Treg amplification with a CD28 superagonistic monoclonal antibody (CD28SA) reduces brain damage in murine cerebral ischemia. METHODS: Cerebral ischemia was induced by coagulation of the distal middle cerebral artery or by 60 minutes filament occlusion of the proximal middle cerebral artery in C57BL6 mice. 150 µg CD28SA was injected intraperitoneally 3 or 6 hours after ischemia onset. Outcome was determined by infarct volumetry and behavioral testing. Brain-infiltrating leukocyte subpopulations were analyzed by flow cytometry and immunohistochemistry 3 and 7 days after middle cerebral artery occlusion. RESULTS: CD28SA reduced infarct size in both models and attenuated functional deficit 7 days after stroke induction. Mice treated with CD28SA increased numbers of Treg in spleen and brain. Tregs were functionally active and migrated into the brain where they accumulated and proliferated in the peri-infarct area. More than 60% of brain infiltrating Treg produced interleukin-10 in CD28SA compared with 30% in control. CONCLUSIONS: In vivo expansion and amplification of Treg by CD28SA attenuates the inflammatory response and improves outcome after experimental stroke.

Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist.

The CD28 superagonist (CD28SA) TGN1412 was administered to humans as an agent that can selectively activate and expand regulatory T cells but resulted in uncontrolled T cell activation accompanied by cytokine storm. The molecular mechanisms that underlie this uncontrolled T cell activation are unclear. Physiological activation of T cells leads to upregulation of not only activation molecules but also inhibitory receptors such as PD-1. We hypothesized that the uncontrolled activation of CD28SA-stimulated T cells is due to both the enhanced expression of activation molecules and the lack of or reduced inhibitory signals. In this study, we show that anti-CD3 antibody-stimulated human T cells undergo time-limited controlled DNA synthesis, proliferation and interleukin-2 secretion, accompanied by PD-1 expression. In contrast, CD28SA-activated T cells demonstrate uncontrolled activation parameters including enhanced expression of LFA-1 and CCR5 but fail to express PD-1 on the cell surface. We demonstrate the functional relevance of the lack of PD-1 mediated regulatory mechanism in CD28SA-stimulated T cells. Our findings provide a molecular explanation for the dysregulated activation of CD28SA-stimulated T cells and also highlight the potential for the use of differential expression of PD-1 as a biomarker of safety for T cell immunostimulatory biologics.

Human regulatory T cells are selectively activated by low-dose application of the CD28 superagonist TGN1412/TAB08.

CD28 superagonists (CD28SAs) are potent T-cell-activating monoclonal antibodies (mAbs). In contrast to their benign behavior and marked therapeutic efficacy as activators of regulatory T (Treg) cells in preclinical rodent models, a phase I trial of the human CD28SA TGN1412 (now called TAB08) in 2006 resulted in a life-threatening cytokine release syndrome (CRS). We studied TAB08-mediated Treg-cell activation in a recently developed in vitro system of human PBMCs, which also reproduces the CRS experienced by the healthy volunteers. We show that just as in rodents, CD28SAs are potent activators and expanders of Treg cells from healthy donors and rheumatoid arthritis patients, even under effective blockade of pro-inflammatory cytokine release by a corticosteroid. Moreover, CD28SA titration identifies a dose range where pro-inflammatory cytokine secretion from conventional T cells is absent while appreciable Treg-cell activation is maintained. Finally, we report that low-dose application of TAB08 to healthy volunteers results in dose-dependent systemic release of the Treg-cell signature cytokine IL-10 in the absence of the pro-inflammatory factors associated with the CRS of the 2006 TGN1412 study. These results demonstrate the potential of appropriately dosed CD28SA and corticosteroid comedication to mobilize human Treg cells for the treatment of autoimmune and inflammatory conditions.

T-cell co-stimulatory blockade in transplantation: two steps forward one step back!

INTRODUCTION: The concern about nephrotoxicity with calcineurin inhibitors led to the search of novel agents for immunosuppression. Based on the requirement of T-cell co-stimulatory signals to fully activated naïve T cells, it became clear that blocking these pathways could be an appealing therapeutic target. However, some unexpected findings were noticed in the recent clinical trials of belatacept, including a higher rate of rejection, which warranted further investigation with some interesting concepts emerging from the bench. AREAS COVERED: This article aims to review the literature of the B7:CD28 co-stimulatory blockade in transplantation, including the basic immunology behind its development, clinical application and potential limitations. EXPERT OPINION: Targeting co-stimulatory pathways were found to be much more complex than initially anticipated due to the interplay between not only various co-stimulatory pathways but also various co-inhibitory ones. In addition, co-stimulatory signals have different roles in diverse immune cell types. Therefore, targeting CD28 ligands with cytotoxic T lymphocyte antigen-4 (CTLA4)-Ig may have some deleterious effects, including the inhibition of regulatory T cells, blockade of co-inhibitory signals (CTLA4) and promotion of Th17 cells. Co-stimulatory independence of memory T cells was another unforeseen limitation. Learning how to better integrate co-stimulatory targeting with other immunosuppressive agents will be critical for the improvement of long-term graft survival.