Cameron J. Turtle

Comprehensive assessment of T-cell receptor β-chain diversity in αβ T cells

The adaptive immune system uses several strategies to generate a repertoire of T- and B-cell antigen receptors with sufficient diversity to recognize the universe of potential pathogens. In alphabeta T cells, which primarily recognize peptide antigens presented by major histocompatibility complex molecules, most of this receptor diversity is contained within the third complementarity-determining region (CDR3) of the T-cell receptor (TCR) alpha and beta chains. Although it has been estimated that the adaptive immune system can generate up to 10(16) distinct alphabeta pairs, direct assessment of TCR CDR3 diversity has not proved amenable to standard capillary electrophoresis-based DNA sequencing. We developed a novel experimental and computational approach to measure TCR CDR3 diversity based on single-molecule DNA sequencing, and used this approach to determine the CDR3 sequence in millions of rearranged TCRbeta genes from T cells of 2 adults. We find that total TCRbeta receptor diversity is at least 4-fold higher than previous estimates, and the diversity in the subset of CD45RO(+) antigen-experienced alphabeta T cells is at least 10-fold higher than previous estimates. These methods should prove valuable for assessment of alphabeta T-cell repertoire diversity after hematopoietic cell transplantation, in states of congenital or acquired immunodeficiency, and during normal aging.

CD19 CAR–T cells of defined CD4+:CD8+ composition in adult B cell ALL patients

BACKGROUND T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells. METHODS We conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. RESULTS The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. CONCLUSION Immunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival. TRIAL REGISTRATION ClinicalTrials.gov NCT01865617. FUNDING R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

Overlap and Effective Size of the Human CD8+ T Cell Receptor Repertoire

Deep sequencing of the T cell receptor repertoires of seven healthy adults reveals that the adaptive immune system is far less diverse than expected and the person-to-person overlap is thousands of times larger. Not So Diverse After All You never know what nasty microbe lurks around the corner. To guard against these potential foes, our immune cells produce a vast variety of antibody and T cell receptor (TCR) shapes ready to recognize these pathogens. This diversity is manufactured by gene rearrangement, with fragments from the so-called V, D, and J gene groups joined together to form an array of sequences, much as colored beads can be arranged on a string to make many combinations. With several choices for each of the V, D, and J fragments, supplemented with a few random nucleotides inserted at the junctions, an astronomical number of different arrangements can theoretically be created. Originally, this was thought to be a random process, but Robins et al., by high-throughput genome sequencing of seven people, show that the generation of immune diversity is actually selective, creating an unexpectedly small assortment of TCRs that is similar in different people. By analyzing blood samples containing millions of T cells from healthy donors and sequencing the TCRs from each donor’s set of naïve and memory T cells, the authors revealed that the sequences of the TCRβ subunits in each subject are not randomly distributed. Instead, certain D and J segments preferentially associated with each other. The number of nucleotides inserted at junction sites between the segments was also smaller than expected. As a result, the actual repertoire of each person’s T cells is a fraction of that predicted by a computer model assuming random rearrangement, and the overlap between donors’ T cells is several orders of magnitude greater. If the rearrangements were truly random, two unrelated adults would only be expected to share on average five TCRβ sequences, of a total 3 million, but the overlap turned out to be more than 10,000 sequences. The authors also overturn another assumption in the field. It had been thought that a random set of V-D-J combinations was generated and then, by deletion and selection in the thymus of cells carrying less useful combinations, only a subset matured, forming the final immune repertoire. Instead, the authors show that the T cell repertoire is limited and biased from the time of the original V-D-J rearrangement during cell development. Now that deep sequencing of these key immune regions in individual genomes is achievable, we will be able to compare TCR sequences and antibodies in healthy individuals with those of patients with autoimmune disorders, or of transplant recipients suffering from graft-versus-host disease. The results may help to understand these illnesses and to look for ways to modify patients’ T cell repertoires for treatment or prevention of autoimmunity or to enhance compatibility between transplant donors and recipients. Diversity in T lymphocyte antigen receptors is generated by somatic rearrangement of T cell receptor (TCR) genes and is concentrated within the third complementarity-determining region 3 (CDR3) of each chain of the TCR heterodimer. We sequenced the CDR3 regions from millions of rearranged TCR β chain genes in naïve and memory CD8+ T cells of seven adults. The CDR3 sequence repertoire realized in each individual is strongly biased toward specific Vβ-Jβ pair utilization, dominated by sequences containing few inserted nucleotides, and drawn from a defined subset comprising less than 0.1% of the estimated 5 × 1011 possible sequences. Surprisingly, the overlap in the naïve CD8+ CDR3 sequence repertoires of any two of the individuals is ~7000-fold larger than predicted and appears to be independent of the degree of human leukocyte antigen matching.

The B-cell tumor–associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor

Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells.

Immunotherapy of non-Hodgkin's lymphoma with a defined ratio of CD8 + and CD4 + CD19-specific chimeric antigen receptor-modified T cells

A CD19 chimeric antigen receptor–modified T cell product with defined composition has potent antitumor activity. Standardizing the CAR assembly line Chimeric antigen receptor (CAR)–modified T cells are engineered to recognize specific tumor antigens. They have shown promising results in clinical trials, primarily in leukemia so far, but it has been difficult to predict therapeutic efficacy and toxicity for individual patients. To address this issue, Turtle et al. treated non-Hodgkin’s lymphoma patients with CAR-T cells prepared from strictly defined subsets. By carefully controlling the ratio of CD4 to CD8 T cells, the authors were able to identify some of the treatment characteristics that correlate with therapeutic response and toxicity, including the role of the drug regimen used for lymphodepletion before CAR-T cell treatment. CD19-specific chimeric antigen receptor (CAR)–modified T cells have antitumor activity in B cell malignancies, but factors that affect toxicity and efficacy have been difficult to define because of differences in lymphodepletion and heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 CD4+/CD8+ ratio of CAR-T cells to 32 adults with relapsed and/or refractory B cell non-Hodgkin’s lymphoma after cyclophosphamide (Cy)–based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had increased CAR-T cell expansion and persistence, and higher response rates [50% complete remission (CR), 72% overall response rate (ORR)] than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR). The CR rate in patients treated with Cy/Flu at the maximally tolerated dose was 64% (82% ORR; n = 11). Cy/Flu minimized the effects of an immune response to the murine single-chain variable fragment component of the CAR, which limited CAR-T cell expansion and clinical efficacy in patients who received Cy-based lymphodepletion without Flu. Severe cytokine release syndrome (sCRS) and grade ≥3 neurotoxicity were observed in 13 and 28% of all patients, respectively. Serum biomarkers, one day after CAR-T cell infusion, correlated with subsequent sCRS and neurotoxicity. Immunotherapy with CD19 CAR-T cells in a defined CD4+/CD8+ ratio allowed identification of correlative factors for CAR-T cell expansion, persistence, and toxicity, and facilitated optimization of lymphodepletion that improved disease response and overall and progression-free survival.

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.

Single-unit dominance after double-unit umbilical cord blood transplantation coincides with a specific CD8+ T-cell response against the nonengrafted unit.

We investigated the potential role of an immune reaction in mediating the dominant engraftment of 1 cord blood unit in 14 patients who received a double-unit cord blood transplantation (CBT). In 10 patients, dominant engraftment of a single donor unit emerged by day 28 after CBT. In 9 of these 10 patients, a significant subset of CD8(+) CD45RO(+/-)CCR7(-) T cells, present in peripheral blood mononuclear cells and derived from the engrafting cord blood unit, produced interferon-gamma (IFN-gamma) in response to the nonengrafting unit. No significant population of IFN-gamma-secreting cells was detectable when posttransplantation peripheral blood mononuclear cells were stimulated against cells from the engrafted unit (P < .001) or from a random human leukocyte antigen disparate third party (P = .003). Three patients maintained persistent mixed chimerism after CBT, and no significant IFN-gamma-secreting cells were detected after similar stimulations in these patients (P < .005). Our data provide the first direct evidence in human double-unit CBT recipients that immune rejection mediated by effector CD8(+) T cells developing after CBT from naive precursors is responsible for the failure of 1 unit to engraft. Future investigations based on these findings may result in strategies to predict a dominant unit and enhance graft-versus-leukemia effect.

A Distinct Subset of Self-Renewing Human Memory CD8+ T Cells Survives Cytotoxic Chemotherapy

The mechanisms that maintain human T cell memory during normal and perturbed homeostasis are not fully understood. The repeated induction of profound lymphocytopenia in patients undergoing multiple cycles of cytotoxic chemotherapy infrequently results in severe infections with viruses controlled by memory T cells, suggesting that some memory T cells survive chemotherapy and restore immunity. Here, we identified a distinct subpopulation of memory CD8(+) T cells with the ability to rapidly efflux and survive exposure to chemotherapy drugs in vitro and in vivo. T cells with high efflux capacity shared expression of molecules with hematopoietic stem cells, were quiescent in nonlymphocytopenic individuals, and were induced to proliferate in patients rendered lymphocytopenic after chemotherapy. Effluxing T cells differentiated into noneffluxing subsets in response to antigen stimulation and inflammatory signals, thereby contributing to repopulation of memory cells after chemotherapy.

Generation of CD19-chimeric antigen receptor modified CD8+ T cells derived from virus-specific central memory T cells

The adoptive transfer of donor T cells that have been genetically modified to recognize leukemia could prevent or treat leukemia relapse after allogeneic HSCT (allo-HSCT). However, adoptive therapy after allo-HSCT should be performed with T cells that have a defined endogenous TCR specificity to avoid GVHD. Ideally, T cells selected for genetic modification would also have the capacity to persist in vivo to ensure leukemia eradication. Here, we provide a strategy for deriving virus-specific T cells from CD45RA(-)CD62L(+)CD8(+) central memory T (T(CM)) cells purified from donor blood with clinical grade reagents, and redirect their specificity to the B-cell lineage marker CD19 through lentiviral transfer of a gene encoding a CD19-chimeric Ag receptor (CAR). Virus-specific T(CM) were selectively transduced by exposure to the CD19 CAR lentivirus after peptide stimulation, and bi-specific cells were subsequently enriched to high purity using MHC streptamers. Activation of bi-specific T cells through the CAR or the virus-specific TCR elicited phosphorylation of downstream signaling molecules with similar kinetics, and induced comparable cytokine secretion, proliferation, and lytic activity. These studies identify a strategy for tumor-specific therapy with CAR-modified T cells after allo-HSCT, and for comparative studies of CAR and TCR signaling.

Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy.

Administration of lymphodepletion chemotherapy followed by CD19-specific chimeric antigen receptor (CAR)-modified T cells is a remarkably effective approach to treating patients with relapsed and refractory CD19(+) B-cell malignancies. We treated 7 patients with B-cell acute lymphoblastic leukemia (B-ALL) harboring rearrangement of the mixed lineage leukemia (MLL) gene with CD19 CAR-T cells. All patients achieved complete remission (CR) in the bone marrow by flow cytometry after CD19 CAR-T-cell therapy; however, within 1 month of CAR-T-cell infusion, 2 of the patients developed acute myeloid leukemia (AML) that was clonally related to their B-ALL, a novel mechanism of CD19-negative immune escape. These reports have implications for the management of patients with relapsed and refractory MLL-B-ALL who receive CD19 CAR-T-cell therapy.