Philip D. Greenberg

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.

Characterization of circulating T cells specific for tumor- associated antigens in melanoma patients

We identified circulating CD8+ T-cell populations specific for the tumor-associated antigens (TAAs) MART-1 (27-35) or tyrosinase (368-376) in six of eleven patients with metastatic melanoma using peptide/HLA-A*0201 tetramers. These TAA-specific populations were of two phenotypically distinct types: one, typical for memory/effector T cells; the other, a previously undescribed phenotype expressing both naive and effector cell markers. This latter type represented more than 2% of the total CD8+ T cells in one patient, permitting detailed phenotypic and functional analysis. Although these cells have many of the hallmarks of effector T cells, they were functionally unresponsive, unable to directly lyse melanoma target cells or produce cytokines in response to mitogens. In contrast, CD8+ T cells from the same patient were able to lyse EBV-pulsed target cells and showed robust allogeneic responses. Thus, the clonally expanded TAA-specific population seems to have been selectively rendered anergic in vivo. Peptide stimulation of the TAA-specific T-cell populations in other patients failed to induce substantial upregulation of CD69 expression, indicating that these cells may also have functional defects, leading to blunted activation responses. These data demonstrate that systemic TAA-specific T-cell responses can develop de novo in cancer patients, but that antigen-specific unresponsiveness may explain why such cells are unable to control tumor growth.

Adoptive T cell therapy of tumors: mechanisms operative in the recognition and elimination of tumor cells.

Publisher Summary This chapter attempts to identify the principles and issues elucidated in animal models that may provide insights for the development of effective approaches to modulate T cell functions to promote the eradication of human tumors. The importance of T cells in surveillance against the outgrowth of tumor cells is probably best demonstrated with virally transformed tumors, such as EBV-induced B cell lymphomas, which occur with markedly increased frequency in transplant patients specifically depleted of T cells, and with UV-induced squamous cell cancers that occur with high frequency in renal transplant patients. Second, the reasons for the growth of presumably immunogenic tumors in immunocompetent hosts have become more apparent with an improved understanding of the function of T cell subsets and the requirements for the induction and expression of T cell responses. The earlier perceptions that cytotoxic T cells recognize foreign proteins integrally inserted in the cell membrane had focused efforts for more than a decade on the use of the powerful new monoclonal antibody technology combined with biochemical purification techniques to isolate tumor antigens expressed on the cell membrane. However, analyses of the requirements for recognition of virally infected cells by CD8 + cytotoxic T cells demonstrated that Class I-restricted T c do not recognize integral membrane proteins but rather recognize intracellular proteins that are synthesized in the cytoplasm. Tumors containing unique or mutated and potentially immunogenic cellular proteins may selectively or predominantly elicit only CD4 + or CD8 + T cell responses because the antigen is adequately presented in the context of only one MHC molecule, and in this setting only T cells from the subset restricted to that MHC molecule appear effective in tumor therapy.

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.

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.

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.

Functional Impairment of CD8+ T Cells by Regulatory T Cells during Persistent Retroviral Infection

The establishment of viral persistence generally requires evasion of the host CD8(+) T cell response. Here we describe a form of evasion wherein the CD8(+) T cells are fully capable of recognizing their cognate antigen but their effector functions are suppressed by regulatory T cells. Virus-specific CD8(+) T cells adoptively transferred into mice persistently infected with Friend virus proliferated and appeared activated, but failed to produce IFNgamma or reduce virus loads. Cotransfer experiments revealed that a subpopulation of CD4(+) T cells from persistently infected mice suppressed IFNgamma production by the CD8(+) T cells. Treatment of persistently infected mice with anti-GITR antibody to ameliorate suppression by regulatory T cells significantly improved IFNgamma production by transferred CD8(+) T cells and allowed a significant reduction in viral loads. The results indicate that CD4(+) regulatory T cells contribute to viral persistence and demonstrate an immunotherapy for treating chronic retroviral infections.

Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

The IL-2 receptor promotes lymphocyte proliferation and induction of the c-myc, bcl-2, and bcl-x genes through the trans-activation domain of Stat5

Studies assessing the role of Stat5 in the IL-2 proliferative signal have produced contradictory, and thus inconclusive, results. One factor confounding many of these studies is the ability of IL-2R to deliver redundant mitogenic signals from different cytoplasmic tyrosines on the IL-2R β-chain (IL-2Rβ). Therefore, to assess the role of Stat5 in mitogenic signaling independent of any redundant signals, all cytoplasmic tyrosines were deleted from IL-2Rβ except for Tyr510, the most potent Stat5-activating site. This deletion mutant retained the ability to induce Stat5 activation and proliferation in the T cell line CTLL-2 and the pro-B cell line BA/F3. A set of point mutations at or near Tyr510 that variably compromised Stat5 activation also compromised the proliferative signal and revealed a quantitative correlation between the magnitude of Stat5 activation and proliferation. Proliferative signaling by a receptor mutant with a weak Stat5 activating site could be rescued by overexpression of wt Stat5a or b. Additionally, the ability of this receptor mutant to induce c-myc, bcl-x, and bcl-2 was enhanced by overexpression of wt Stat5. By contrast, overexpression of a version of Stat5a lacking the C-terminal trans-activation domain inhibited the induction of these genes and cell proliferation. Thus, Stat5 is a critical component of the proliferative signal from Tyr510 of the IL-2R and regulates expression of both mitogenic and survival genes through its trans-activation domain.

Interleukin-15 rescues tolerant CD8 + T cells for use in adoptive immunotherapy of established tumors

CD8+ T cells can mediate eradication of established tumors, and strategies to amplify tumor-reactive T-cell numbers by immunization or ex vivo expansion followed by adoptive transfer are currently being explored in individuals with cancer. Generating effective CD8+ T cell–mediated responses to tumors is often impeded by T-cell tolerance to relevant tumor antigens, as most of these antigens are also expressed in normal tissues. We examined whether such tolerant T cells could be rescued and functionally restored for use in therapy of established tumors. We used a transgenic T-cell receptor (TCR) mouse model in which peripheral CD8+ T cells specific for a candidate tumor antigen also expressed in liver are tolerant, failing to proliferate or secrete interleukin (IL)-2 in response to antigen. Molecular and cellular analysis showed that these tolerant T cells expressed the IL-15 receptor α chain, and could be induced to proliferate in vitro in response to exogenous IL-15. Such proliferation abrogated tolerance and the rescued cells became effective in treating leukemia. Therefore, high-affinity CD8+ T cells are not necessarily deleted by encounter with self-antigen in the periphery, and can potentially be rescued and expanded for use in tumor immunotherapy.