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 alpha 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.     

Human breast carcinoma patients develop clonable oncofetal antigen-specific effector and regulatory T lymphocytes.

Oncofetal Ag (OFA) is a 44-kDa glycoprotein expressed during early to mid-gestation fetal development and re-expressed as a surface Ag by tumor cells soon after transformation. The Ag is detectable on all types of human and rodent tumors tested, but is undetectable on normal cells. In experimental animals it is autoimmunogenic and induces potentially protective T cell responses both after experimental immunization and during tumor development subsequent to carcinogenic insult. To determine whether this tumor-associated Ag is also immunogenic for human T lymphocytes, breast carcinoma patients' peripheral blood mononuclear leucocytes were stimulated in vitro with autologous tumor cells in the presence of IL-2, gamma-IFN, and IL-6 for 2 wk. The tumor-reactive cells were then restimulated and cloned by limiting dilution, and the clones were analyzed. We established 24, 19, 11, and 16 tumor-reactive clones from the four respective patients. Of those, 4, 6, 4, and 7, respectively, proliferated specifically to purified OFA. Both CD4 and CD8 OFA-specific clones were established, which responded equally well to purified OFA or 32- to 44-kDa immature laminin receptor protein. All were CD3+, TCR-alpha beta+. All CD4 clones secreted gamma-IFN, but neither secreted IL-4 nor IL-10. Both IFN-gamma-secreting cytotoxic CD8 clones and IL-10-secreting inhibitory CD8 clones were established. Thus, during human cancer development, the same types of OFA-specific effector and regulatory T cells are induced as during murine T lymphomagenesis.     

Activated NKT cells and NK cells render T cells resistant to myeloid-derived suppressor cells and result in an effective adoptive cellular therapy against breast cancer in the FVBN202 transgenic mouse

Attempts to cure breast cancer by adoptive cellular therapy (ACT) have not been successful. This is primarily due to the presence of tumor-induced immune-suppressive mechanisms as well as the failure of tumor-reactive T cells to provide long-term memory responses in vivo. To address these clinically important challenges, we developed an ex vivo protocol for the expansion of tumor-reactive immune cells obtained from tumor-bearing animals prior to or after local radiation therapy. We used an Ag-free protocol that included bryostatin 1/ionomycin and sequential common γ-chain cytokines (IL-7/IL-15 + IL-2). The proposed protocol expanded tumor-reactive T cells as well as activated non-T cells, including NKT cells, NK cells, and IFN-γ-producing killer dendritic cells. Antitumor efficacy of T cells depended on the presence of non-T cells. The effector non-T cells also rendered T cells resistant to myeloid-derived suppressor cells. Radiation therapy altered phenotypic distribution and differentiation of T cells as well as their ability to generate central memory T cells. ACT by means of the expanded cells protected animals from tumor challenge and generated long-term memory responses against the tumor, provided that leukocytes were derived from tumor-bearing animals prior to radiation therapy. The ex vivo protocol was also able to expand HER-2/neu-specific T cells derived from the PBMC of a single patient with breast carcinoma. These data suggest that the proposed ACT protocol should be studied further in breast cancer patients.     

Generation of melanoma-specific, cytotoxic CD4(+) T helper 2 cells: requirement of both HLA-DR15 and Fas antigens on melanomas for their lysis by Th2 cells

Recognition of melanoma antigens by HLA class-II-restricted CD4(+) T lymphocytes has been investigated. Two cytotoxic CD4(+) T cell lines were established by stimulating PBLs from a melanoma patient with either parental or IFN-gamma-transduced autologous tumor cells. These T cells secreted IL-4, but not IL-2, IFN-gamma, or TNF-beta, in response to the autologous melanoma cells, suggesting that they belong to the Th2 subtype. Their cytotoxicity was directed against the IFN-gamma-transduced melanoma cells and was HLA-DR-restricted. The autologous and two allogeneic IFN-gamma-modified melanoma cell lines shared melanoma antigen(s) presented in the context of HLA-DR15. HLA-DR15(+) nonmelanoma cells were resistant targets indicating that the shared antigen(s) is melanoma associated. Parental autologous and HLA-DR-matched allogeneic melanoma cell lines, displaying low levels of HLA-DR antigens, induced Th2 proliferation and cytokine release, but were insensitive to lysis prior to upregulation of HLA-DR and Fas antigens by IFN-gamma. Cytolysis was inhibited by anti-HLA-DR and by anti-Fas antibodies, suggesting that the cytolysis is mediated via the Fas pathway. While small amounts of HLA-DR15 molecules on melanoma cells are sufficient for Th2 proliferation and cytokine release, higher amounts of HLA-DR15 and the expression of Fas are required for CD4(+)-mediated lysis.     

Abrogation of SRC homology region 2 domain-containing phosphatase 1 in tumor-specific T cells improves efficacy of adoptive immunotherapy by enhancing the effector function and accumulation of short-lived effector T cells in vivo

T cell expression of inhibitory proteins can be a critical component for the regulation of immunopathology owing to self-reactivity or potentially exuberant responses to pathogens, but it may also limit T cell responses to some malignancies, particularly if the tumor Ag being targeted is a self-protein. We found that the abrogation of Src homology region 2 domain-containing phosphatase-1 (SHP-1) in tumor-reactive CD8(+) T cells improves the therapeutic outcome of adoptive immunotherapy in a mouse model of disseminated leukemia, with benefit observed in therapy employing transfer of CD8(+) T cells alone or in the context of also providing supplemental IL-2. SHP-1(-/-) and SHP-1(+/+) effector T cells were expanded in vitro for immunotherapy. Following transfer in vivo, the SHP-1(-/-) effector T cells exhibited enhanced short-term accumulation, followed by greater contraction, and they ultimately formed similar numbers of long-lived, functional memory cells. The increased therapeutic effectiveness of SHP-1(-/-) effector cells was also observed in recipients that expressed the tumor Ag as a self-antigen in the liver, without evidence of inducing autoimmune toxicity. SHP-1(-/-) effector CD8(+) T cells expressed higher levels of eomesodermin, which correlated with enhanced lysis of tumor cells. Furthermore, reduction of SHP-1 expression in tumor-reactive effector T cells by retroviral transduction with vectors that express SHP-1-specific small interfering RNA, a translatable strategy, also exhibited enhanced antitumor activity in vivo. These studies suggest that abrogating SHP-1 in effector T cells may improve the efficacy of tumor elimination by T cell therapy without affecting the ability of the effector cells to persist and provide a long-term response.     

Preassociation of IL-15 with IL-15R alpha-IgG1-Fc enhances its activity on proliferation of NK and CD8+/CD44high T cells and its antitumor action

In the induction of an immune response, IL-15Ralpha on APCs transpresents IL-15 to NK and CD8(+)/CD44(high) T cells that express the IL-2/15Rbeta and gammac subunits only. In this study, we show data mimicking this transpresentation by using IL-15 preassociated with a chimeric protein that is comprised of the extracellular domain of murine IL-15Ralpha and the Fc portion of human IgG1. When tested in vitro, IL-15Ralpha-IgG1-Fc strongly increased the IL-15-mediated proliferation of murine NK and CD8(+)/CD44(high) T cells. The effect of IL-15Ralpha-IgG1-Fc was dependent on the presence of both IgG1-Fc and IL-15Ralpha. When injected into mice, IL-15Ralpha-IgG1-Fc enhanced the capacity of IL-15 to expand the number of NK and CD8(+)/CD44(high) T cells. The effect on cell numbers in vivo also depended on Fc receptor binding because reduced expansion was observed in FcRgamma(-/-) mice. NK cells cultured in IL-15/IL-15Ralpha-IgG1-Fc complex gained cytotoxic activity toward a number of NK-sensitive targets. When mice bearing the NK-sensitive syngeneic tumor B16 were treated, the presence of IL-15Ralpha-IgG1-Fc increased the antitumor activity of IL-15. Thus, a preassociation with IL-15Ralpha-IgG1-Fc enhances the activities of IL-15 in vivo and in vitro that may be useful in the treatment of tumors.     

Effects of distant metastasis and peripheral CA 15-3 on the induction of spontaneous T cell responses in breast cancer patients

Tumor-specific memory T cells are detectable in the bone marrow (BM) of a majority of breast cancer patients. In vitro they can be reactivated to IFN-γ producing, cytotoxic effector cells and reject autologous, xenotransplanted tumors in NOD/SCID mice after specific restimulation with autologous dendritic cells (DC). In this study, we demonstrate the presence of specific tumor-reactive BM memory T cells in altogether 56 out of 129 primarily operated breast cancer patients by short-term IFN-γ EliSpot assays with unstimulated T cells and tumor antigen presenting, autologous DCs. We observed tumor-reactive BM memory T cells predominantly in patients with primarily metastatic disease (P = 0.011) or with increased concentrations of tumor marker CA 15-3 in the peripheral blood (P = 0.004), respectively. Memory T cell reactivity against HLA-A^*0201-restricted peptides from the tumor-associated antigens MUC1, Hpa_16–24 and Hpa_183–191 was also detected particularly in patients with elevated peripheral CA 15-3 concentrations (P < 0.05). Altogether these data indicate that the systemic presence of tumor-derived antigens promotes an induction of tumor-specific cellular immune responses in the human BM.     

Cytotoxic T lymphocytes generated by short-term in vitro TCR stimulation in the presence of IL-4 are therapeutically effective against B16 melanoma

P14 TCR transgenic CD8+ T cells (LCMV gp33-specific) were activated by antigen in the presence of either IL-2 or IL-2+IL-4 to generate effector cytotoxic T lymphocytes (CTLs). The therapeutic effectiveness of such IL-2- or IL-2+IL-4-grown CTLs was tested in mice that had received intravenous inoculations of B16.gp33 melanoma cells 7 days previously. Administration of P14 CTLs activated by antigen +IL-2+IL-4 was significantly more effective at reducing melanoma colony formation in the lung than those grown in the presence of antigen +IL-2. Highly significant improvement in survival was observed with 80% of B16.gp33-inoculated mice showing long-term survival after therapy with 10×10⁶ antigen +IL-2+IL-4-activated P14 CTLs. Similar therapeutic effectiveness of antigen +IL-2+IL-4-activated P14 CTLs against subcutaneously inoculated B16.gp33 melanoma cells was also found. There was significant reduction in P14 CD8+ T cells in the peripheral blood of B16.gp33-inoculated mice than in mice that did not receive B16.gp33 melanoma cells, indicating possible homing of P14 CD8+ T cells to the site of tumor growth or antigen-induced apoptotic cell death. These results may have implications in tumor therapy using CTLs grown ex vivo, especially during early stages of tumor formation. They also support the concept that the therapeutic effectiveness of CTLs can be governed by the cytokine context in which they are activated.     

Expression of macrophage migration inhibitory factor by neuroblastoma leads to the inhibition of antitumor T cell reactivity in vivo

Neuroblastomas and many other solid tumors produce high amounts of macrophage migration inhibitory factor (MIF), which appears to play a role in tumor progression. We found that MIF expression in neuroblastoma inhibits T cell proliferation in vitro, raising the possibility that MIF promotes tumorigenesis, in part, by suppressing antitumor immunity. To examine whether tumor-derived MIF leads to suppression of T cell immunity in vivo, we generated MIF-deficient neuroblastoma cell lines using short hairpin small interfering RNAs (siRNA). The MIF knockdown (MIFKD) AGN2a neuroblastoma cells were more effectively rejected in immune-competent mice than control siRNA-transduced or wild-type AGN2a. However, the increased rejection of MIFKD AGN2a was not observed in T cell-depleted mice. MIFKD tumors had increased infiltration of CD8(+) and CD4(+) T cells, as well as increased numbers of macrophages, dendritic cells, and B cells. Immunization with MIFKD AGN2a cells significantly increased protection against tumor challenge as compared with immunization with wild-type AGN2a, and the increased protection correlated with elevated frequencies of tumor-reactive CD8(+) T cells in the lymphoid tissue of treated animals. Increased numbers of infiltrating tumor-reactive CD8(+) T cells were also observed at the site of tumor vaccination. In vitro, treatment of AGN2a-derived culture supernatants with neutralizing MIF-specific Ab failed to reverse T cell suppressive activity, suggesting that MIF is not directly responsible for the immune suppression in vivo. This supports a model whereby MIF expression in neuroblastoma initiates a pathway that leads to the suppression of T cell immunity in vivo.     

Primed tumor-reactive multifunctional CD62L<Superscript>+</Superscript> human CD8<Superscript>+</Superscript> T cells for immunotherapy

T cell-mediated immunotherapy against malignancies has been shown to be effective for certain types of cancer. However, ex vivo expansion of tumor-reactive T cells has been hindered by the low precursor frequency of such cells, often requiring multiple rounds of stimulation, resulting in full differentiation, loss of homing receptors and potential exhaustion of the expanded T cells. Here, we show that when using highly purified naïve CD8⁺ T cells, a single stimulation with peptide-pulsed, IFNγ/LPS-matured dendritic cells in combination with the sequential use of IL-21, IL-7 and IL-15 is sufficient for extensive expansion of antigen-specific T cells. Short-term expanded T cells were tumor-reactive, multifunctional and retained a central-memory-like phenotype (CD62L⁺, CCR7⁺, CD28⁺). The procedure is highly reproducible and robust as demonstrated for different healthy donors and for cancer patients. Such short-term tumor-antigen-primed, multifunctional T cells may therefore serve as a platform to target different malignancies accessible to immunotherapy.     

Tumor-specific CD4+ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor

This study focuses on the specific CD4+ T cell requirement for optimal induction of cytotoxicity against MHC class II negative autologous tumors (AuTu) collected from patients with various types of cancer at advanced stages. CD4+ T cells were induced in cultures of cancer patients' malignant effusion-associated mononuclear cells with irradiated AuTu (mixed lymphocyte tumor cultures (MLTC)) in the presence of recombinant IL-2 and recombinant IL-7. Tumor-specific CD4+ T cells did not directly recognize the AuTu cells, but there was an MHC class II-restricted cross-priming by autologous dendritic cells (DCs), used as APC. CD8+ CTL, also induced during the MLTC, lysed specifically AuTu cells or DCs pulsed with AuTu peptide extracts (acid wash extracts (AWE)) in an MHC class I-restricted manner. Removal of CD4+ T cells or DCs from the MLTC drastically reduced the CD8+ CTL-mediated cytotoxic response against the AuTu. AWE-pulsed DCs preincubated with autologous CD4+ T cells were able, in the absence of CD4+ T cells, to stimulate CD8+ T cells to lyse autologous tumor targets. Such activated CD8+ T cells produced IL-2, IFN-gamma, TNF-alpha, and GM-CSF. The process of the activation of AWE-pulsed DCs by CD4+ T cells could be inhibited with anti-CD40 ligand mAb. Moreover, the role of CD4+ T cells in activating AWE-pulsed DCs was undertaken by anti-CD40 mAb. Our data demonstrate for the first time in patients with metastatic cancer the essential role of CD4+ Th cell-activated DCs for optimal CD8+ T cell-mediated killing of autologous tumors and provide the basis for the design of novel protocols in cellular adoptive immunotherapy of cancer, utilizing synthetic peptides capable of inducing T cell help in vivo.     

Frequency analysis of tumor-reactive cytotoxic T lymphocytes in peripheral blood of a melanoma patient vaccinated with autologous tumor cells

A limiting-dilution assay was developed and used to determine the frequency of autologous tumor-reactive cytotoxic T lymphocytes (CTL) in peripheral blood of a melanoma patient MZ2, who has been free of detectable disease since several years. In this patient, the frequencies of tumor-reactive CTL spontaneously varied only by a factor of 1.5. After vaccinations with autologous mutagenized and lethally irradiated tumor cells a two- to tenfold increase in frequencies of tumor-reactive CTL was found within the first 2 weeks. Thereafter, CTL frequencies returned to values measured prior to vaccinations. We conclude, that the limiting-dilution assay applied in this study can detect changes in the T cell response to autologous tumor cells. The frequency of tumor-reactive CTL determined with this approach can serve as an immunological parameter for monitoring the T cell response to autologous tumor cells in individual cancer patients receiving tumor cell vaccinations.     

Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells

The important role of tumor-specific cytotoxic CD8⁺ T cells is well defined in the immune control of the tumors, but the role of effector CD4⁺ T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4⁺ T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4⁺ T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8⁺ T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4⁺ T cells and increases FV-specific CD4⁺ T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4⁺ T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4⁺ T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.     

Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apopotosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-γ release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a “per-cell” basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

CTLA-4 blockade by a human MAb enhances the capacity of AML-derived DC to induce T-cell responses against AML cells in an autologous culture system

BACKGROUND: Cells from AML patients can differentiate into the phenotype of DC when cultured with GM-CSF and IL-4. Such cytokine-treated AML-derived DC (AML-DC) can stimulate autologous T cells. In this study we examined whether an anti-CTLA-4 MAb (MDX-010) could enhance the generation of autologous anti-AML T cells. METHODS: MAb MDX-010 was added to AML PBMC cultures in the presence of GM-CSF and IL-4, a previously reported AML-DC culture method of generating anti-AML T cells. T-cell activation and proliferation were examined thereafter. RESULTS: Addition of MDX-010 to GM-CSF/IL-4-conditioned AML-DC cultures induced a mean seven-fold increase in the numbers of autologous T cells compared with cultures without MDX-010 (P < 0.007). T cells stimulated by AML-DC with CTLA-4 blockade were significantly more cytotoxic towards autologous AML cells than those without MDX-010 (42 +/- 23% vs. 26 +/- 15%, E:T ratio of 20). T cells stimulated by AML-DC with CTLA-4 blockade had significantly greater proportions of T cells producing IFN-gamma in response to autologous AML cells than those cultured with AML-DC alone (10.7 +/- 4.7% vs. 4.5 +/- 2.4% for CD4+ IFN-gamma+ CD69+ and 9.8 +/- 4.1% vs. 4 +/- 2.1% for CD8+ IFN-gamma+ CD69+ with or without MDX-010; n = 7; P < 0.007, P < 0.003, respectively). DISCUSSION: CTLA-4 blockade enhances the activity and numbers of AML-reactive T cells that can be stimulated by autologous AML-DC and may enhance the efficacy of adoptive immunotherapy of AML.     

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.     

Synergy between interleukin-2 and prothymosin α for the increased generation of cytotoxic T lymphocytes against autologous human carcinomas

 Peripheral blood mononuclear cells (PBMC) from cancer patients were cultured in vitro with irradiated autologous tumor cells isolated from malignant effusions (mixed lymphocyte tumor cultures, MLTC) and low-dose (50 IU/ml) recombinant interleukin-2 (IL-2). The combination of IL-2 and prothymosin α (ProTα) resulted in a greater PBMC-induced response to the autologous tumor than that brought about by IL-2 alone. In particular, ProTα specifically enhanced the CD4⁺ T-cell-mediated proliferation against the autologous tumor. CD4⁺ T cells seemed to recognize tumor antigens presented by HLA-DR molecules expressed on the autologous monocytes, since preincubation of the latter with an anti-HLA-DR monoclonal antibody (mAb) abrogated the response. In addition, MLTC set up with IL-2 and ProTα also generated more MHC-class-I-restricted cytotoxic T lymphocytes (CTL) against the autologous tumor than did MLTC set up with IL-2 alone. The MLTC-induced CTL contained high levels of cytoplasmic perforin and their development was strictly dependent on the presence of both autologous CD4⁺ T cells and monocytes. In the absence of either population there was a strong impairment of both proliferative and cytotoxic responses which was not restored by the presence of ProTα. In contrast, when both cell populations were present, ProTα exerted optimal enhancement of CD4⁺ T cell proliferation, which was associated with potentiated CTL responses. Our data emphasize the role of ProTα for the enhancement of IL-2-induced CTL responses against autologous tumor cells. Such responses require collaborative interactions between CD4⁺, CD8⁺ T cells and monocytes as antigen-presenting cells. Our data are relevant for adoptive immunotherapeutic settings utilizing IL-2 and ProTα-induced autologous-tumor-specific CTL. Received: 2 March 2000 / Accepted: 1 June 2000     

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.