BATF3 programs CD8+ T cell memory.

Antiviral CD8+ T cell responses are characterized by an initial activation/priming of T lymphocytes followed by a massive proliferation, subset differentiation, population contraction and the development of a stable memory pool. The transcription factor BATF3 has been shown to play a central role in the development of conventional dendritic cells, which in turn are critical for optimal priming of CD8+ T cells. Here we show that BATF3 was expressed transiently within the first days after T cell priming and had long-lasting T cell-intrinsic effects. T cells that lacked Batf3 showed normal expansion and differentiation, yet succumbed to an aggravated contraction and had a diminished memory response. Vice versa, BATF3 overexpression in CD8+ T cells promoted their survival and transition to memory. Mechanistically, BATF3 regulated T cell apoptosis and longevity via the proapoptotic factor BIM. By programing CD8+ T cell survival and memory, BATF3 is a promising molecule to optimize adoptive T cell therapy in patients.

SLAMF7-CAR T cells eliminate myeloma and confer selective fratricide of SLAMF7+ normal lymphocytes.

SLAMF7 is under intense investigation as a target for immunotherapy in multiple myeloma. In this study, we redirected the specificity of T cells to SLAMF7 through expression of a chimeric antigen receptor (CAR) derived from the huLuc63 antibody (elotuzumab) and demonstrate that SLAMF7-CAR T cells prepared from patients and healthy donors confer potent antimyeloma reactivity. We confirmed uniform, high-level expression of SLAMF7 on malignant plasma cells in previously untreated and in relapsed/refractory (R/R) myeloma patients who had received previous treatment with proteasome inhibitors and immunomodulatory drugs. Consequently, SLAMF7-CAR T cells conferred rapid cytolysis of previously untreated and R/R primary myeloma cells in vitro. In addition, a single administration of SLAMF7-CAR T cells led to resolution of medullary and extramedullary myeloma manifestations in a murine xenograft model in vivo. SLAMF7 is expressed on a fraction of normal lymphocytes, including subsets of natural killer (NK) cells, T cells, and B cells. After modification with the SLAMF7-CAR, both CD8+ and CD4+ T cells rapidly acquired and maintained a SLAMF7- phenotype and could be readily expanded to therapeutically relevant cell doses. We analyzed the recognition of normal lymphocytes by SLAMF7-CAR T cells and show that they induce selective fratricide of SLAMF7+/high NK cells, CD4+ and CD8+ T cells, and B cells. Importantly, however, the fratricide conferred by SLAMF7-CAR T cells spares the SLAMF7-/low fraction in each cell subset and preserves functional lymphocytes, including virus-specific T cells. In aggregate, our data illustrate the potential use of SLAMF7-CAR T-cell therapy as an effective treatment against multiple myeloma and provide novel insights into the consequences of targeting SLAMF7 for the normal lymphocyte compartment.

Interrupting CD28 costimulation before antigen rechallenge affects CD8(+) T-cell expansion and effector functions during secondary response in mice.

The role of CD28-mediated costimulation in secondary CD8(+) T-cell responses remains controversial. Here, we have used two tools - blocking mouse anti-mouse CD28-specific antibodies and inducible CD28-deleting mice - to obtain definitive answers in mice infected with ovalbumin-secreting Listeria monocytogenes. We report that both blockade and global deletion of CD28 reveal its requirement for full clonal expansion and effector functions such as degranulation and IFN-γ production during the secondary immune response. In contrast, cell-intrinsic deletion of CD28 in transferred TCR-transgenic CD8(+) T cells before primary infection leads to impaired clonal expansion but an increase in cells able to express effector functions in both primary and secondary responses. We suggest that the proliferation-impaired CD8(+) T cells respond to CD28-dependent help from their environment by enhanced functional differentiation. Finally, we report that cell-intrinsic deletion of CD28 after the peak of the primary response does not affect the establishment, maintenance, or recall of long-term memory. Thus, if given sufficient time, the progeny of primed CD8(+) T cells adapt to the absence of this costimulator.

Minicircle-Based Engineering of Chimeric Antigen Receptor (CAR) T Cells.

Plasmid DNA is being used as a pharmaceutical agent in vaccination, as well as a basic substance and starting material in gene and cell therapy, and viral vector production. Since the uncontrolled expression of backbone sequences present in such plasmids and the dissemination of antibiotic resistance genes may have profound detrimental effects, an important goal in vector development was to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle (MC) DNA. The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform enabling a close-to-random profile of genomic integration. In combination, the MC platform greatly enhances SB transposition and transgene integration resulting in higher numbers of stably modified target cells. We have recently developed a strategy for MC-based SB transposition of chimeric antigen receptor (CAR) transgenes that enable improved transposition rates compared to conventional plasmids and rapid manufacturing of therapeutic CAR T cell doses (Monjezi et al. 2016). This advance enables manufacturing CAR T cells in a virus-free process that relies on SB-mediated transposition from MC DNA to accomplish gene-transfer. Advantages of this approach include a strong safety profile due to the nature of the MC itself and the genomic insertion pattern of MC-derived CAR transposons. In addition, stable transposition and high-level CAR transgene expression, as well as easy and reproducible handling, make MCs a preferred vector source for gene-transfer in advanced cellular and gene therapy. In this chapter, we will review our experience in MC-based CAR T cell engineering and discuss our recent advances in MC manufacturing to accelerate both pre-clinical and clinical implementation.

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.

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.

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.

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

The costimulatory receptor CD28 and IL-4Ralpha 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-4Ralpha 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-4Ralpha 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-4Ralpha 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.

On-target restoration of a split T cell-engaging antibody for precision immunotherapy.

T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.

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.

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.

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 p110delta(D910A/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.

Inhibition of IL-4/IL-13 does not enhance the efficacy of allergen immunotherapy in murine allergic airway inflammation.

BACKGROUND: Successful allergen-specific immunotherapy (SIT) is associated with reduced Th2 cytokine production and the induction of IL-10-producing regulatory T cells. To improve treatment efficacy, we investigated the impact of an IL-4/IL-13 inhibitor during SIT. METHODS: BALB/c mice were sensitized intranasally with ovalbumin (OVA) for 4 weeks. Subsequently, they were subjected to intranasal SIT, with OVA being administered at doses increasing from 1 mug to 1 mg over 3 weeks with or without an IL-4/IL-13 inhibitor. Serum OVA-specific antibodies were measured and bronchoalveolar lavage (BAL) fluids were checked for airway eosinophilia. Subsequently, lung tissue was examined histologically for inflammatory infiltrates. Cytokines were detected in BAL fluids and spleen cell cultures. Furthermore, CD4 CD25 double-positive spleen T cells were checked for intracellular IL-10 production by flow cytometry. RESULTS: OVA sensitization resulted in persistent IgE synthesis and an eosinophil-rich allergic airway inflammation combined with increased IL-4 and IL-5 levels. Therefore, intranasal SIT could efficiently reverse the allergic phenotype. This was associated with decreased IL-4 and IL-5 levels, and increased IL-10 levels in BAL fluids as well as increased amounts of IL-10-producing CD25+ regulatory T cells. However, mice treated with the IL-4/IL-13 inhibitor during SIT did not produce significantly different results . CONCLUSION: The use of an IL-4/IL-13 inhibitor as an adjuvant for SIT did not enhance anti-allergic effects. Thus, the observed reversal of Th2 responses during SIT may not be the keystone for successful therapy, but rather other factors, e.g. IL-10-producing regulatory T cells, may be crucial.