Tea Gogishvili

Chimeric antigen receptor-modified T cells derived from defined CD8 + and CD4 + subsets confer superior antitumor reactivity in vivo

Adoptive T-cell therapy with gene-modified T cells expressing a tumor-reactive T-cell receptor or chimeric antigen receptor (CAR) is a rapidly growing field of translational medicine and has shown success in the treatment of B-cell malignancies and solid tumors. In all reported trials, patients have received T-cell products comprising random compositions of CD4+ and CD8+ naive and memory T cells, meaning that each patient received a different therapeutic agent. This variation may have influenced the efficacy of T-cell therapy, and complicates comparison of outcomes between different patients and across trials. We analyzed CD19 CAR-expressing effector T cells derived from different subsets (CD4+/CD8+ naive, central memory, effector memory). T cells derived from each of the subsets were efficiently transduced and expanded, but showed clear differences in effector function and proliferation in vitro and in vivo. Combining the most potent CD4+ and CD8+ CAR-expressing subsets, resulted in synergistic antitumor effects in vivo. We show that CAR-T-cell products generated from defined T-cell subsets can provide uniform potency compared with products derived from unselected T cells that vary in phenotypic composition. These findings have important implications for the formulation of T-cell products for adoptive therapies.

Rapid Regulatory T-Cell Response Prevents Cytokine Storm in CD28 Superagonist Treated Mice

Superagonistic CD28-specific monoclonal antibodies (CD28SA) are highly effective activators of regulatory T-cells (Treg cells) in rats, but a first-in-man trial of the human CD28SA TGN1412 resulted in an unexpected cytokine release syndrome. Using a novel mouse anti-mouse CD28SA, we re-investigate the relationship between Treg activation and systemic cytokine release. Treg activation by CD28SA was highly efficient but depended on paracrine IL-2 from CD28SA-stimulated conventional T-cells. Systemic cytokine levels were innocuous, but depletion of Treg cells prior to CD28SA stimulation led to systemic release of proinflammatory cytokines, indicating that in rodents, Treg cells effectively suppress the inflammatory response. Since the human volunteers of the TGN1412 study were not protected by this mechanism, we also tested whether corticosteroid prophylaxis would be compatible with CD28SA induced Treg activation. We show that neither the expansion nor the functional activation of Treg cells is affected by high-dose dexamethasone sufficient to control systemic cytokine release. Our findings warn that preclinical testing of activating biologicals in rodents may miss cytokine release syndromes due to the rapid and efficacious response of the rodent Treg compartment, and suggest that polyclonal Treg activation is feasible in the presence of antiphlogistic corticosteroid prophylaxis.

Regulatory T cells facilitate the nuclear accumulation of inducible cAMP early repressor (ICER) and suppress nuclear factor of activated T cell c1 (NFATc1)

Inducible cAMP early repressor (ICER) is a transcriptional repressor, which, because of alternate promoter use, is generated from the 3′ region of the cAMP response modulator (Crem) gene. Its expression and nuclear occurrence are elevated by high cAMP levels in naturally occurring regulatory T cells (nTregs). Using two mouse models, we demonstrate that nTregs control the cellular localization of ICER/CREM, and thereby inhibit IL-2 synthesis in conventional CD4+ T cells. Ablation of nTregs in depletion of regulatory T-cell (DEREG) mice resulted in cytosolic localization of ICER/CREM and increased IL-2 synthesis upon stimulation. Direct contacts between nTregs and conventional CD4+ T cells led to nuclear accumulation of ICER/CREM and suppression of IL-2 synthesis on administration of CD28 superagonistic (CD28SA) Ab. In a similar way, nTregs communicated with B cells and induced the cAMP-driven nuclear localization of ICER/CREM. High levels of ICER suppressed the induction of nuclear factor of activated T cell c1 (Nfatc1) gene in T cells whose inducible Nfatc1 P1 promoter bears two highly conserved cAMP-responsive elements to which ICER/CREM can bind. These findings suggest that nTregs suppress T-cell responses by the cAMP-dependent nuclear accumulation of ICER/CREM and inhibition of NFATc1 and IL-2 induction.

Cell-intrinsic and -extrinsic control of Treg-cell homeostasis and function revealed by induced CD28 deletion

While the requirement for CD28 and its ligands for the generation and function of “natural” (n)Treg cells is well established, it has not been possible yet to investigate cell‐intrinsic effects after interrupted CD28 expression. Here, we demonstrate a selective loss of Treg cells after disruption of the CD28 gene. The decline in Treg‐cell number was accompanied by reduced homeostatic proliferation, probably due to lack of costimulation during self‐antigen recognition, and by impaired Treg‐cell function including downregulation of CTLA‐4. The decline in Treg‐cell number was unaffected by thymectomy or by the presence of CD28 expressing T cells within the same animal, indicating that impairment of peripheral homeostasis and function of nTreg cells by CD28 deletion is cell‐intrinsic. In contrast, downregulation of CD25, the α chain of the IL‐2R, did not occur in the presence of WT T cells, indicating that its expression does not depend on CD28 signals in cis.

Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors.

Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation. Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs). We analyzed genomic distribution of SB and LV integrations and show that a significantly higher proportion of MC-derived CAR transposons compared with LV integrants had occurred outside of highly expressed and cancer-related genes into genomic safe harbor loci that are not expected to cause mutagenesis or genotoxicity. CD19-CAR T cells engineered with our enhanced SB approach conferred potent reactivity in vitro and eradicated lymphoma in a xenograft model in vivo. Intriguingly, electroporation of SB MCs is substantially more effective and less toxic compared with conventional plasmids, and enables cost-effective rapid preparation of therapeutic CAR T-cell doses. This approach sets a new standard in advanced cellular and gene therapy and will accelerate and increase the availability of CAR T-cell therapy to treat hematologic malignancies.

CD28 and IL-4: two heavyweights controlling the balance between immunity and inflammation

The costimulatory receptor CD28 and IL-4Rα-containing cytokine receptors play key roles in controlling the size and quality of pathogen-specific immune responses. Thus, CD28-mediated costimulation is needed for effective primary T-cell expansion and for the generation and activation of regulatory T-cells (Treg cells), which protect from immunopathology. Similarly, IL-4Rα signals are required for alternative activation of macrophages, which counteract inflammation by type 1 responses. Furthermore, immune modulation by CD28 and IL-4 is interconnected through the promotion of IL-4 producing T-helper 2 cells by CD28 signals. Using conditionally IL-4Rα and CD28 deleting mice, as well as monoclonal antibodies, which block or stimulate CD28, or mAb that deplete Treg cells, we have studied the roles of CD28 and IL-4Rα in experimental mouse models of virus (influenza), intracellular bacteria (L. monocytogenes, M. tuberculosis), and parasite infections (T. congolense, L. major). We observed that in some, but not all settings, Treg cells and type 2 immune deviation, including activation of alternative macrophages can be manipulated to protect the host either from infection or from immunopathology with an overall beneficial outcome. Furthermore, we provide direct evidence that secondary CD8 T-cell responses to i.c. bacteria are dependent on CD28-mediated costimulation.

Proliferative signals mediated by CD28 superagonists require the exchange factor Vav1 but not phosphoinositide 3-kinase in primary peripheral T cells†

Almost all responses of naive T cells require co‐stimulation, i.e. engagement of the clonotypic TCR with relevant antigen/MHC and the co‐stimulatory molecule CD28. How CD28 contributes to T‐cell proliferation remains poorly understood, with widely conflicting reports existing which may reflect different methods of co‐ligating receptors. Some CD28 mAb, however, can stimulate T‐cell proliferation without the need for TCR co‐ligation, and thus provide unique tools to dissect proliferative signals mediated through CD28 alone. Using primary peripheral T cells from CD28‐transgenic mice, we show that both the YMNM and Lck‐binding motifs, but not the Itk‐binding motif, in CD28 are required for proliferation. Given that the YMNM motif recruits both phosphoinositide 3‐kinase (PI3K) and the exchange factor Vav1, we investigated the role of these two molecules in CD28‐mediated proliferation. In p110δD910A/D910A transgenic T cells, which are defective in PI3K activation following CD28 ligation, proliferation was comparable to that in wild‐type cells. By contrast, T‐cell proliferation was abolished in Vav1−/− cells. Although we did not address the role of Grb2 in CD28 signalling, these results indicate that CD28 can mediate Lck‐ and Vav1‐dependent proliferative signals independently of PI3K.

Sequential induction of effector function, tissue migration and cell death during polyclonal activation of mouse regulatory T-cells.

The ability of CD4+Foxp3+ regulatory T-cells (Treg) to produce interleukin (IL)-10 is important for the limitation of inflammation at environmental interfaces like colon or lung. Under steady state conditions, however, few Tregs produce IL-10 ex vivo. To investigate the origin and fate of IL-10 producing Tregs we used a superagonistic mouse anti-mouse CD28 mAb (CD28SA) for polyclonal in vivo stimulation of Tregs, which not only led to their numeric expansion but also to a dramatic increase in IL-10 production. IL-10 secreting Tregs strongly upregulated surface receptors associated with suppressive function as compared to non-producing Tregs. Furthermore, polyclonally expanding Tregs shifted their migration receptor pattern after activation from a CCR7+CCR5− lymph node-seeking to a CCR7−CCR5+ inflammation-seeking phenotype, explaining the preferential recruitment of IL-10 producers to sites of ongoing immune responses. Finally, we observed that IL-10 producing Tregs from CD28SA stimulated mice were more apoptosis-prone in vitro than their IL-10 negative counterparts. These findings support a model where prolonged activation of Tregs results in terminal differentiation towards an IL-10 producing effector phenotype associated with a limited lifespan, implicating built-in termination of immunosuppression.

Interruption of CD28-mediated costimulation during allergen challenge protects mice from allergic airway disease.

BACKGROUND Allergic asthma is a T(H)2-promoted hyperreactivity with an immediate, IgE, and mast cell-dependent response followed by eosinophil-dominated inflammation and airway obstruction. OBJECTIVE Because costimulation by CD28 is essential for T(H)2 but not T(H)1 responses, we investigated the effect of selective interference with this pathway in mice using the models of ovalbumin and house dust mite-induced airway inflammation. METHODS To study the role of CD28 in the effector phase of allergic airway inflammation, we developed an inducibly CD28-deleting mouse strain or alternatively used a CD28 ligand-binding site-specific mouse anti-mouse mAb blocking CD28 engagement. RESULTS We show that even after systemic sensitization to the allergen, interruption of CD28-mediated costimulation is highly effective in preventing airway inflammation during challenge. In addition to improving airway resistance and histopathologic presentation and reducing inflammatory infiltrates, antibody treatment during allergen challenge resulted in a marked relative increase in regulatory T-cell numbers among the CD4 T-cell subset of the challenged lung. CONCLUSION Selective interference with CD28-mediated costimulation during allergen exposure might be an attractive therapeutic concept for allergic asthma.

Panobinostat induces CD38 upregulation and augments the anti-myeloma efficacy of daratumumab

To the editor: The anti-CD38 monoclonal antibody (mAb) daratumumab is effective in multiple myeloma (MM) and is increasingly being used at first relapse in combination with bortezomib/dexamethasone or lenalidomide/dexamethasone, which are capable of inducing complete responses in a notable