Optimization of anti-CD19 CAR T cell production for treatment of patients with chronic lymphocytic leukemia.

T cells expressing anti-CD19 chimeric antigen receptors (CARs) have activity against chronic lymphocytic leukemia (CLL), but complete response rates range from 18% to 29%, so improvement is needed. Peripheral blood mononuclear cells (PBMCs) of CLL patients often contain high levels of CLL cells that can interfere with CAR T cell production, and T cells from CLL patients are prone to exhaustion and other functional defects. We previously developed an anti-CD19 CAR designated Hu19-CD828Z. Hu19-CD828Z has a binding domain derived from a fully human antibody and a CD28 costimulatory domain. We aimed to develop an optimized process for producing Hu19-CD828Z-expressing T cells (Hu19-CAR T) from PBMC of CLL patients. We determined that supplementing Hu19-CAR-T cultures with interleukin (IL)-7 + IL-15 had advantages over using IL-2, including greater accumulation of Hu19-CAR T cells during in vitro proliferation assays. We determined that positive selection with anti-CD4 and anti-CD8 magnetic beads was the optimal method of T cell purification because this method resulted in high T cell purity. We determined that anti-CD3/CD28 paramagnetic beads were the optimal T cell activation reagent. Finally, we developed a current good manufacturing practices-compliant clinical-scale protocol for producing Hu19-CAR T from PBMC of CLL patients. These Hu19-CAR T exhibited a full range of in vitro functions and eliminated leukemia from mice.

TGFß prevents IgE-mediated allergic disease by restraining T follicular helper 2 differentiation.

Allergic diseases are common, affecting more than 20% of the population. Genetic variants in the TGFß pathway are strongly associated with atopy. To interrogate the mechanisms underlying this association, we examined patients and mice with Loeys-Dietz syndrome (LDS) who harbor missense mutations in the kinase domain of TGFΒR1/2. We demonstrate that LDS mutations lead to reduced TGFß signaling and elevated total and allergen-specific IgE, despite the presence of wild-type T regulatory cells in a chimera model. Germinal center activity was enhanced in LDS and characterized by a selective increase in type 2 follicular helper T cells (TFH2). Expression of Pik3cg was increased in LDS TFH cells and associated with reduced levels of the transcriptional repressor SnoN. PI3Kγ/mTOR signaling in LDS naïve CD4+ T cells was elevated after T cell receptor cross-linking, and pharmacologic inhibition of PI3Kγ or mTOR prevented exaggerated TFH2 and antigen-specific IgE responses after oral antigen exposure in an adoptive transfer model. Naïve CD4+ T cells from nonsyndromic allergic individuals also displayed decreased TGFß signaling, suggesting that our mechanistic discoveries may be broadly relevant to allergic patients in general. Thus, TGFß plays a conserved, T cell-intrinsic, and nonredundant role in restraining TFH2 development via the PI3Kγ/mTOR pathway and thereby protects against allergic disease.

Rapid anti-myeloma activity by T cells expressing an anti-BCMA CAR with a human heavy-chain-only antigen-binding domain.

Multiple myeloma (MM) is a rarely curable malignancy of plasma cells. MM expresses B cell maturation antigen (BCMA). We developed a fully human anti-BCMA chimeric antigen receptor (CAR) with a heavy-chain-only antigen-recognition domain, a 4-1BB domain, and a CD3ζ domain. The CAR was designated FHVH33-CD8BBZ. We conducted the first-in-humans clinical trial of T cells expressing FHVH33-CD8BBZ (FHVH-T). Twenty-five patients with relapsed MM were treated. The stringent complete response rate (sCR) was 52%. Median progression-free survival (PFS) was 78 weeks. Of 24 evaluable patients, 6 (25%) had a maximum cytokine-release syndrome (CRS) grade of 3; no patients had CRS of greater than grade 3. Most anti-MM activity occurred within 2-4 weeks of FHVH-T infusion as shown by decreases in the rapidly changing MM markers serum free light chains, urine light chains, and bone marrow plasma cells. Blood CAR+ cell levels peaked during the time that MM elimination was occurring, between 7 and 15 days after FHVH-T infusion. C-C chemokine receptor type 7 (CCR7) expression on infusion CD4+ FHVH-T correlated with peak blood FHVH-T levels. Single-cell RNA sequencing revealed a shift toward more differentiated FHVH-T after infusion. Anti-CAR antibody responses were detected in 4 of 12 patients assessed. FHVH-T has powerful, rapid, and durable anti-MM activity.

Epithelial-intrinsic defects in TGFßR signaling drive local allergic inflammation manifesting as eosinophilic esophagitis.

Allergic diseases are a global health challenge. Individuals harboring loss-of-function variants in transforming growth factor-ß receptor (TGFßR) genes have an increased prevalence of allergic disorders, including eosinophilic esophagitis. Allergic diseases typically localize to mucosal barriers, implicating epithelial dysfunction as a cardinal feature of allergic disease. Here, we describe an essential role for TGFß in the control of tissue-specific immune homeostasis that provides mechanistic insight into these clinical associations. Mice expressing a TGFßR1 loss-of-function variant identified in atopic patients spontaneously develop disease that clinically, immunologically, histologically, and transcriptionally recapitulates eosinophilic esophagitis. In vivo and in vitro, TGFßR1 variant-expressing epithelial cells are hyperproliferative, fail to differentiate properly, and overexpress innate proinflammatory mediators, which persist in the absence of lymphocytes or external allergens. Together, our results support the concept that TGFß plays a fundamental, nonredundant, epithelial cell-intrinsic role in controlling tissue-specific allergic inflammation that is independent of its role in adaptive immunity.

Recent developments in understanding the mechanisms of food allergy.

PURPOSE OF REVIEW: The prevalence of food allergy is rising globally. This review will discuss recent discoveries regarding the immunologic mechanisms that drive the initial sensitization and allergic response to food antigens, which may inform prevention and treatment strategies. RECENT FINDINGS: Tolerance to food antigens is antigen-specific and promoted by oral exposure early in life and maternal transfer of immune complexes via breast milk. IgG can inhibit both the initiation and effector phases of allergic responses to food antigens in mice, and high levels of food-specific IgG4 are associated with acquisition of tolerance in humans. Disruption of the skin barrier provides a route for food sensitization through the actions of mast cells, type 2 innate lymphoid cells, and IL-33 signaling. Regulatory T cells (Tregs) promote acquisition of oral tolerance, although defects in circulating allergen-specific Tregs are not evident in children with established food allergy. Certain microbes can offer protection against the development of IgE and food allergic responses, while dysbiosis increases susceptibility to food allergy. SUMMARY: Tolerance to food antigens is antigen-specific and is promoted by oral exposure early in life, maternal transfer of immune complexes, food-specific IgG, Tregs, an intact skin barrier, and a healthy microbiome.

Genetic evidence for the role of transforming growth factor-ß in atopic phenotypes.

New evidence in humans and mice supports a role for transforming growth factor-ß (TGF-ß) in the initiation and effector phases of allergic disease, as well as in consequent tissue dysfunction. This pleiotropic cytokine can affect T cell activation and differentiation and B cell immunoglobulin class switching following initial encounter with an allergen. TGF-ß can also act on mast cells during an acute allergic episode to modulate the strength of the response, in addition to driving tissue remodeling following damage caused by an allergic attack. Accordingly, genetic disorders leading to altered TGF-ß signaling can result in increased rates of allergic disease.

Identification and analysis of peanut-specific effector T and regulatory T cells in children allergic and tolerant to peanut.

BACKGROUND: Peanut allergy (PA) is potentially life-threatening and generally persists for life. Recent data suggest the skin might be an important route of initial sensitization to peanut, whereas early oral exposure to peanut is protective. In mice regulatory T (Treg) cells are central to the development of food tolerance, but their contribution to the pathogenesis of food allergy in human subjects is less clear. OBJECTIVE: We sought to quantify and phenotype CD4+ peanut-specific effector T (ps-Teff) cells and peanut-specific regulatory T (ps-Treg) cells in children with and without PA or PS. METHODS: ps-Teff and ps-Treg cells were identified from peripheral blood of children with PA, children with PS, and nonsensitized/nonallergic (NA) school-aged children and 1-year-old infants based on upregulation of CD154 or CD137, respectively, after stimulation with peanut extract. Expression of cytokines and homing receptors was evaluated by using flow cytometry. Methylation at the forkhead box protein 3 (FOXP3) locus was measured as a marker of Treg cell stability. RESULTS: Differential upregulation of CD154 and CD137 efficiently distinguished ps-Teff and ps-Treg cells. A greater percentage of ps-Teff cells from infants with PA and infants with PS expressed the skin-homing molecule cutaneous lymphocyte antigen, suggesting activation after exposure through the skin, compared with NA infants. Although ps-Teff cells in both school-aged and infant children with PA produced primarily TH2 cytokines, a TH1-skewed antipeanut response was seen only in NA school-aged children. The frequency, homing receptor expression, and stability of ps-Treg cells in infants and school-aged children were similar, regardless of allergic status. CONCLUSIONS: Exposure to peanut through the skin can prime the development of TH2 ps-Teff cells, which promote sensitization to peanut, despite the presence of normal numbers of ps-Treg cells.

Distinct modes of antigen presentation promote the formation, differentiation, and activity of foxp3+ regulatory T cells in vivo.

How the formation and activity of CD4(+)Foxp3(+) regulatory T cells (Tregs) are shaped by TCR recognition of the diverse array of peptide:MHC complexes that can be generated from self-antigens and/or foreign Ags in vivo remains poorly understood. We show that a self-peptide with low (but not high) stimulatory potency promotes thymic Treg formation and can induce conventional CD4(+) T cells in the periphery to become Tregs that express different levels of the transcription factor Helios according to anatomical location. When Tregs generated in response to this self-peptide subsequently encountered the same peptide derived instead from influenza virus in the lung-draining lymph nodes of infected mice, they proliferated, acquired a T-bet(+)CXCR3(+) phenotype, and suppressed the antiviral effector T cell response in the lungs. However, these self-antigen-selected Tregs were unable to suppress the antiviral immune response based on recognition of the peptide as a self-antigen rather than a viral Ag. Notably, when expressed in a more immunostimulatory form, the self-peptide inhibited the formation of T-bet(+)CXCR3(+) Tregs in response to viral Ag, and Ag-expressing B cells from these mice induced Treg division without upregulation of CXCR3. These studies show that a weakly immunostimulatory self-peptide can induce thymic and peripheral Foxp3(+) Treg formation but is unable to activate self-antigen-selected Tregs to modulate an antiviral immune response. Moreover, a strongly immunostimulatory self-peptide expressed by B cells induced Tregs to proliferate without acquiring an effector phenotype that allows trafficking from the draining lymph node to the lungs and, thereby, prevented the Tregs from suppressing the antiviral immune response.

The role of T-cell receptor recognition of peptide:MHC complexes in the formation and activity of Foxp3⁺ regulatory T cells.

Foxp3(+) regulatory T (Treg) cells are required to prevent the immune system from spontaneously mounting a severe autoaggressive lymphoproliferative disease and can modulate immune responses in a variety of settings, including infections. In this review, we describe studies that use transgenic mice to determine how signals through the T-cell receptor (TCR) contribute to the development, differentiation, and activity of Treg cells in in vivo settings. By varying the amount and quality of the self-peptide recognized by an autoreactive TCR, we have shown that the interplay between autoreactive thymocyte deletion and Treg cell formation leads to a Treg cell repertoire that is biased toward low abundance agonist self-peptides. In an autoimmune disease setting, we have demonstrated that diverse TCR specificities can be required in order for Treg cells to prevent disease in a mouse model of autoimmune inflammatory arthritis. Lastly, we have shown that Treg cells initially selected based on specificity for a self-peptide can be activated by TCR recognition of a viral peptide, and that they can acquire a specialized phenotype and suppress antiviral effector cell activity at the site of infection. These studies provide insights into the pivotal role that TCR specificity plays in the formation and activity of Treg cells.

Strength of TCR signal from self-peptide modulates autoreactive thymocyte deletion and Foxp3(+) Treg-cell formation.

Autoreactive CD4(+) CD8(-) (CD4SP) thymocytes can be subjected to deletion when they encounter self-peptide during their development, but they can also undergo selection to become CD4SPFoxp3(+) Treg cells. We have analyzed the relationship between these distinct developmental fates using mice in which signals transmitted by the TCR have been attenuated by mutation of a critical tyrosine residue of the adapter protein SLP-76. In mice containing polyclonal TCR repertoires, the mutation caused increased frequencies of CD4SPFoxp3(+) thymocytes. CD4SP thymocytes expressing TCR Vß-chains that are subjected to deletion by endogenous retroviral superantigens were also present at increased frequencies, particularly among Foxp3(+) thymocytes. In transgenic mice in which CD4SP thymocytes expressing an autoreactive TCR undergo both deletion and Treg-cell formation in response to a defined self-peptide, SLP-76 mutation abrogated deletion of autoreactive CD4SP thymocytes. Notably, Foxp3(+) Treg-cell formation still occurred, albeit with a reduced efficiency, and the mutation was also associated with decreased Nur77 expression by the autoreactive CD4SP thymocytes. These studies provide evidence that the strength of the TCR signal can play a direct role in directing the extent of both thymocyte deletion and Treg-cell differentiation, and suggest that distinct TCR signaling thresholds and/or pathways can promote CD4SP thymocyte deletion versus Treg-cell formation.