Stanley R. Riddell

Reconstitution of Cellular Immunity against Cytomegalovirus in Recipients of Allogeneic Bone Marrow by Transfer of T-Cell Clones from the Donor

BACKGROUND Cytomegalovirus (CMV) disease in immunocompromised patients correlates with a deficiency of CD8+ cytotoxic T lymphocytes specific for CMV. We evaluated the safety and immunologic effects of immunotherapy with clones of these lymphocytes in recipients of allogeneic bone marrow transplants. METHODS Clones of CD8+ cytotoxic T cells specific for CMV proteins were isolated from the blood of bone marrow donors. Fourteen patients each received four intravenous infusions of these clones from their donors beginning 30 to 40 days after marrow transplantation. The reconstitution of cellular immunity against CMV was monitored before and during the period of infusions and for up to 12 weeks after the final infusion. The rearranged genes encoding the T-cell receptor served as markers in evaluating the persistence of the transferred T cells. RESULTS No toxic effects related to the infusions were observed. Cytotoxic T cells specific for CMV were reconstituted in all patients. In vitro measurements showed that cytotoxic activity against CMV was significantly increased (P < 0.001) after the infusions in 11 patients who were deficient in such activity before therapy. The level of activity achieved after the infusions was similar to that measured in the donors. Analysis of rearranged T-cell-receptor genes in T cells obtained from two recipients indicated that the transferred clones persisted for at least 12 weeks. Cytotoxic-T-cell activity declined in patients deficient in CD4+ T-helper cells specific for CMV, suggesting that helper-T-cell function is needed for the persistence of transferred CD8+ T cells. Neither CMV viremia nor CMV disease developed in any of the 14 patients. CONCLUSIONS The transfer of CMV-specific clones of CD8+ T cells derived from the bone marrow donor is a safe and effective way to reconstitute cellular immunity against CMV after allogeneic marrow transplantation.

Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: In vivo persistence, migration, and antitumor effect of transferred T cells

Adoptive T cell therapy, involving the ex vivo selection and expansion of antigen-specific T cell clones, provides a means of augmenting antigen-specific immunity without the in vivo constraints that can accompany vaccine-based strategies. A phase I study was performed to evaluate the safety, in vivo persistence, and efficacy of adoptively transferred CD8+ T cell clones targeting the tumor-associated antigens, MART1/MelanA and gp100 for the treatment of patients with metastatic melanoma. Four infusions of autologous T cell clones were administered, the first without IL-2 and subsequent infusions with low-dose IL-2 (at 0.25, 0.50, and 1.0 × 106 units/m2 twice daily for the second, third, and fourth infusions, respectively). Forty-three infusions of MART1/MelanA-specific or gp100-specific CD8+ T cell clones were administered to 10 patients. No serious toxicity was observed. We demonstrate that the adoptively transferred T cell clones persist in vivo in response to low-dose IL-2, preferentially localize to tumor sites and mediate an antigen-specific immune response characterized by the elimination of antigen-positive tumor cells, regression of individual metastases, and minor, mixed or stable responses in 8 of 10 patients with refractory, metastatic disease for up to 21 mo.

Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells.

NKG2D is an activating receptor that stimulates innate immune responses by natural killer cells upon engagement by MIC ligands, which are induced by cellular stress. Because NKG2D is also present on most CD8αβ T cells, it may modulate antigen-specific T cell responses, depending on whether MIC molecules—distant homologs of major histocompatibility complex (MHC) class I with no function in antigen presentation—are induced on the surface of pathogen-infected cells. We found that infection by cytomegalovirus (CMV) resulted in substantial increases in MIC on cultured fibroblast and endothelial cells and was associated with induced MIC expression in interstitial pneumonia. MIC engagement of NKG2D potently augmented T cell antigen receptor (TCR)-dependent cytolytic and cytokine responses by CMV-specific CD28− CD8αβ T cells. This function overcame viral interference with MHC class I antigen presentation. Combined triggering of TCR-CD3 complexes and NKG2D induced interleukin 2 production and T cell proliferation. Thus NKG2D functioned as a costimulatory receptor that can substitute for CD28.

Adoptive transfer of effector CD8+ T cells derived from central memory cells establishes persistent T cell memory in primates.

The adoptive transfer of antigen-specific T cells that have been expanded ex vivo is being actively pursued to treat infections and malignancy in humans. The T cell populations that are available for adoptive immunotherapy include both effector memory and central memory cells, and these differ in phenotype, function, and homing. The efficacy of adoptive immunotherapy requires that transferred T cells persist in vivo, but identifying T cells that can reproducibly survive in vivo after they have been numerically expanded by in vitro culture has proven difficult. Here we show that in macaques, antigen-specific CD8(+) T cell clones derived from central memory T cells, but not effector memory T cells, persisted long-term in vivo, reacquired phenotypic and functional properties of memory T cells, and occupied memory T cell niches. These results demonstrate that clonally derived CD8+ T cells isolated from central memory T cells are distinct from those derived from effector memory T cells and retain an intrinsic capacity that enables them to survive after adoptive transfer and revert to the memory cell pool. These results could have significant implications for the selection of T cells to expand or to engineer for adoptive immunotherapy of human infections or malignancy.

T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients

The introduction and expression of genes in somatic cells is an innovative therapy for correcting genetic deficiency diseases and augmenting immune function. A potential obstacle to gene therapy is the elimination of such gene–modified cells by an immune response to novel protein products of the introduced genes. We are conducting an immunotherapy trial in which individuals seropositive for human immunodeficiency virus (HIV) receive CD8+ HIV–specific cytotoxic T cells modified by retroviral transduction to express a gene permitting positive and negative selection. However, five of six subjects developed cytotoxic T–lymphocyte responses specific for the novel protein and eliminated the transduced cytotoxic T cells. The rejection of genetically modified cells by these immunocompromised hosts suggests that strategies to render gene–modified cells less susceptible to host immune surveillance will be required for successful gene therapy of immunocompetent hosts.

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.

Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells

Adoptive immunotherapy with T cells expressing a tumor-specific chimeric T-cell receptor is a promising approach to cancer therapy that has not previously been explored for the treatment of lymphoma in human subjects. We report the results of a proof-of-concept clinical trial in which patients with relapsed or refractory indolent B-cell lymphoma or mantle cell lymphoma were treated with autologous T cells genetically modified by electroporation with a vector plasmid encoding a CD20-specific chimeric T-cell receptor and neomycin resistance gene. Transfected cells were immunophenotypically similar to CD8(+) effector cells and showed CD20-specific cytotoxicity in vitro. Seven patients received a total of 20 T-cell infusions, with minimal toxicities. Modified T cells persisted in vivo 1 to 3 weeks in the first 3 patients, who received T cells produced by limiting dilution methods, but persisted 5 to 9 weeks in the next 4 patients who received T cells produced in bulk cultures followed by 14 days of low-dose subcutaneous interleukin-2 (IL-2) injections. Of the 7 treated patients, 2 maintained a previous complete response, 1 achieved a partial response, and 4 had stable disease. These results show the safety, feasibility, and potential antitumor activity of adoptive T-cell therapy using this approach. This trial was registered at www.clinicaltrials.gov as #NCT00012207.

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.

The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells.

Antigen-specific T cell clones are useful reagents for studies of the fine specificity of antigen recognition and of potential therapeutic use in adoptive immunotherapy for human viral and malignant diseases. Culture methods which require antigen and APC for stimulation can be problematic for the generation and long-term growth of human virus and tumor-specific T cells. We have developed an alternative culture method using monoclonal antibodies to T cell activation molecules, CD3 and CD28, as stimulation to efficiently grow CD4+ and CD8+ antigen-specific T cells from single progenitors and expand T cell clones in long-term culture. This method alleviates the requirement for large amounts of viral or tumor antigens and MHC compatible APC to sustain the growth of virus and tumor-specific T cell clones, and, as demonstrated for CD8+ CMV-specific cytotoxic T cells, overcomes the difficulties cloning CD8+ T cells using virally infected cells as antigen-presenting cells. T cell clones generated and maintained with monoclonal antibody stimulation are rapidly expanded and retain antigen-specific responses after 3 months in culture, suggesting this approach may prove useful for growing large numbers of antigen-specific T cell clones for cellular immunotherapy.

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.