Papillomavirus-specific CD4+ T cells exhibit reduced STAT-5 signaling and altered cytokine profiles in patients with recurrent respiratory papillomatosis

Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6) or HPV-11. Specific HLA-DR haplotypes DRB1*01:02 and DRB1*03:01 are associated with the development of RRP, disease severity, and Th2-like responses to HPV early proteins. Th1-like responses to HPV proteins have been shown to be protective in animal models. Therefore, we investigated the hypothesis that RRP patients have dysfunctional Th1-like, HPV-specific T cell responses. Using MHC class II tetramers, we identified immunogenic peptides within HPV-11 early proteins. Two distinct peptides (E6(113-132) and E2(1-20)) contained DRB1*01:02- or DRB1*03:01-restricted epitopes, respectively. An additional peptide (E2(281-300)) contained an epitope presented by both alleles. Peptide binding, tetramer, and proliferation assays identified minimal epitopes within these peptides. These epitopes elicited E2/E6-specific CD4(+) T cell responses in RRP patients and healthy control subjects, allowing the isolation of HPV-specific T cell lines using tetramers. The cytokine profiles and STAT signaling of these tetramer-positive T cells were measured to compare the polarization and responsiveness of HPV-specific T cells from patients with RRP and healthy subjects. HPV-specific IFN-γ secretion was substantially lower in T cells from RRP patients. HPV-specific IL-13 secretion was seen at modest levels in T cells from RRP patients and was absent in T cells from healthy control subjects. HPV-specific T cells from RRP patients exhibited reduced STAT-5 phosphorylation and reduced IL-2 secretion, suggesting anergy. Levels of STAT-5 phosphorylation and IFN-γ secretion could be improved through addition of IL-2 to HPV-specific T cell lines from RRP patients. Therapeutic vaccination or interventions aimed at restoring Th1-like cytokine responses to HPV proteins and reversing anergy could improve clinical outcomes for RRP patients.     

Low-dose cyclophosphamide administered as daily or single dose enhances the antitumor effects of a therapeutic HPV vaccine

Although therapeutic HPV vaccines are able to elicit systemic HPV-specific immunity, clinical responses have not always correlated with levels of vaccine-induced CD8⁺ T cells in human clinical trials. This observed discrepancy may be attributable to an immunosuppressive tumor microenvironment in which the CD8⁺ T cells are recruited. Regulatory T cells (Tregs) are cells that can dampen cytotoxic CD8⁺ T-cell function. Cyclophosphamide (CTX) is a systemic chemotherapeutic agent, which can eradicate immune cells, including inhibitory Tregs. The optimal dose and schedule of CTX administration in combination with immunotherapy to eliminate the Treg population without adversely affecting vaccine-induced T-cell responses is unknown. Therefore, we investigated various dosing and administration schedules of CTX in combination with a therapeutic HPV vaccine in a preclinical tumor model. HPV tumor-bearing mice received either a single preconditioning dose or a daily dose of CTX in combination with the pNGVL4a-CRT/E7(detox) DNA vaccine. Both single and daily dosing of CTX in combination with vaccine had a synergistic antitumor effect as compared to monotherapy alone. The potent antitumor responses were attributed to the reduction in Treg frequency and increased infiltration of HPV16 E7-specific CD8⁺ T cells, which led to higher ratios of CD8⁺/Treg and CD8⁺/CD11b⁺Gr-1⁺ myeloid-derived suppressor cells (MDSCs). There was an observed trend toward decreased vaccine-induced CD8⁺ T-cell frequency with daily dosing of CTX. We recommend a single, preconditioning dose of CTX prior to vaccination due to its efficacy, ease of administration, and reduced cumulative adverse effect on vaccine-induced T cells.     

Human papilloma virus–specific T cells can be generated from naïve T cells for use as an immunotherapeutic strategy for immunocompromised patients

Human papilloma virus (HPV) is a known cause of cervical cancer, squamous cell carcinoma and laryngeal cancer. Although treatments exist for HPV-associated malignancies, patients unresponsive to these therapies have a poor prognosis. Recent findings from vaccine studies suggest that T-cell immunity is essential for disease control. Because Epstein-Barr Virus (EBV)-specific T cells have been highly successful in treating or preventing EBV-associated tumors, we hypothesized that the development of a manufacturing platform for HPV-specific T cells from healthy donors could be used in a third-party setting to treat patients with high-risk/relapsed HPV-associated cancers. Most protocols for generating virus-specific T cells require prior exposure of the donor to the targeted virus and, because the seroprevalence of high-risk HPV types varies greatly by age and ethnicity, manufacturing of donor-derived HPV-specific T cells has proven challenging. We, therefore, made systematic changes to our current Good Manufacturing Practice (GMP)-compliant protocols to improve antigen presentation, priming and expansion for the manufacture of high-efficacy HPV-specific T cells. Like others, we found that current methodologies fail to expand HPV-specific T cells from most healthy donors. By optimizing dendritic cell maturation and function with lipopolysaccharide (LPS) and interferon (IFN)γ, adding interleukin (IL)-21 during priming and depleting memory T cells, we achieved reliable expansion of T cells specific for oncoproteins E6 and E7 to clinically relevant amounts (mean, 578-fold expansion; n?=?10), which were polyfunctional based on cytokine multiplex analysis. In the third-party setting, such HPV-specific T-cell products might serve as a potent salvage therapy for patients with HPV-associated diseases.     

Immunogenicity of dendritic cells pulsed with MAGE3, Survivin and B-cell maturation antigen mRNA for vaccination of multiple myeloma patients

The introduction of autologous stem cell transplantation (SCT) and novel drugs has improved overall survival in multiple myeloma (MM) patients. However, minimal residual disease (MRD) remains and most patients eventually relapse. Myeloma plasma cells express tumor-associated antigens (TAA), which are interesting targets for immunotherapy. In this phase 1 study, we investigated the safety and immunological effects of TAA-mRNA-loaded dendritic cell (DC) vaccination for treatment for MRD in MM after SCT. Mature monocyte-derived DCs were pulsed with keyhole limpet hemocyanin (KLH) and electroporated with MAGE3, Survivin or B-cell maturation antigen (BCMA) mRNA. Twelve patients were vaccinated three times with intravenous (5–22 × 10⁶ DCs) and intradermal vaccines (4–11 × 10⁶ DCs), at biweekly intervals. Immunological responses were monitored in blood and delayed-type hypersensitivity (DTH) biopsies. All patients developed strong anti-KLH T-cell responses, but not KLH antibodies. In 2 patients, vaccine-specific T cells were detected in DTH biopsies. In one patient, we found MAGE3-specific CD4⁺ and CD8⁺ T cells, and CD3⁺ T cells reactive against BCMA and Survivin. In the other patient, we detected low numbers of MAGE3 and BCMA-reactive CD8⁺ T cells. Vaccination was well tolerated with limited toxicity. These findings illustrate that TAA-mRNA-electroporated mature DCs are capable of inducing TAA-T-cell responses in MM patients after SCT.     

Recombinant IL-21 and anti-CD4 antibodies cooperate in syngeneic neuroblastoma immunotherapy and mediate long-lasting immunity

IL-21 is an immune-enhancing cytokine, which showed promising results in cancer immunotherapy. We previously observed that the administration of anti-CD4 cell-depleting antibody strongly enhanced the anti-tumor effects of an IL-21-engineered neuroblastoma (NB) cell vaccine. Here, we studied the therapeutic effects of a combination of recombinant (r) IL-21 and anti-CD4 monoclonal antibodies (mAb) in a syngeneic model of disseminated NB. Subcutaneous rIL-21 therapy at 0.5 or 1 μg/dose (at days 2, 6, 9, 13 and 15 after NB induction) had a limited effect on NB development. However, coadministration of rIL-21 at the two dose levels and a cell-depleting anti-CD4 mAb cured 28 and 70 % of mice, respectively. Combined immunotherapy was also effective if started 7 days after NB implant, resulting in a 30 % cure rate. Anti-CD4 antibody treatment efficiently depleted CD4⁺ CD25^high Treg cells, but alone had limited impact on NB. Combination immunotherapy by anti-CD4 mAb and rIL-21 induced a CD8⁺ cytotoxic T lymphocyte response, which resulted in tumor eradication and long-lasting immunity. CD4⁺ T cells, which re-populated mice after combination immunotherapy, were required for immunity to NB antigens as indicated by CD4⁺ T cell depletion and re-challenge experiments. In conclusion, these data support a role for regulatory CD4⁺ T cells in a syngeneic NB model and suggest that rIL-21 combined with CD4⁺ T cell depletion reprograms CD4⁺ T cells from immune regulatory to anti-tumor functions. These observations open new perspectives for the use of IL-21-based immunotherapy in conjunction with transient CD4⁺ T cell depletion, in human metastatic NB.     

Intratumoral temozolomide synergizes with immunotherapy in a T cell-dependent fashion

Despite temozolomide (TMZ) treatment, the prognosis for patients with glioblastoma multiforme is still dismal. As dose escalation of TMZ is limited by systemic toxicity, intratumoral delivery emerges as an attractive treatment modality, which may sustain cytotoxic drug concentrations intratumorally and induce immunogenic cell death. Both clinical and experimental gliomas have responded to immunotherapy, but the benefit of simultaneous chemo- and immunotherapy is inadequately studied. Here, we monitored survival of GL261-bearing C57BL/6 mice following a 3-day treatment with either intratumoral TMZ (micro-osmotic pump, 4.2 mg/kg/day) or systemic TMZ (i.p. injections, 50 mg/kg/day) alone, or combined with immunization using GM-CSF secreting GL261 cells. Peripheral and intratumoral leukocytes were analyzed by flow cytometry and immunohistochemistry. Intratumoral TMZ induced higher survival rate than systemic TMZ (45 vs. 8 %). When T cells were depleted following intratumoral TMZ, the therapeutic effect was completely abrogated (0 % survival). Intratumoral TMZ synergistically increased survival rate of immunized mice (from 25 to 83 %), while systemic TMZ failed (0 %). While systemic TMZ induced a transient leukopenia, intratumoral TMZ and immunotherapy sustained the proliferation of CD8⁺ T cells and decreased the number of intratumoral immunosuppressive cells. In conclusion, intratumoral TMZ alone or in combination with immunotherapy could cure glioma-bearing mice, due to attenuation of local immunosuppression and increase in potential effector immune cells.     

A combination trial of vaccine plus ipilimumab in metastatic castration-resistant prostate cancer patients: immune correlates

We recently reported the clinical results of a Phase I trial combining ipilimumab with a vaccine containing transgenes for prostate-specific antigen (PSA) and for a triad of costimulatory molecules (PROSTVAC) in patients with metastatic castration-resistant prostate cancer. Thirty patients were treated with escalating ipilimumab and a fixed dose of vaccine. Of 24 chemotherapy-naïve patients, 58 % had a PSA decline. Combination therapy did not exacerbate the immune-related adverse events associated with ipilimumab. Here, we present updated survival data and an evaluation of 36 immune cell subsets pre- and post-therapy. Peripheral blood mononuclear cells were collected before therapy, at 13 days and at 70 days post-initiation of therapy, and phenotyped by flow cytometry for the subsets of T cells, regulatory T cells, natural killer cells, and myeloid-derived suppressor cells. Associations between overall survival (OS) and immune cell subsets prior to treatment, and the change in a given immune cell subset 70 days post-initiation of therapy, were evaluated. The median OS was 2.63 years (1.77–3.45). There were trends toward associations for longer OS and certain immune cell subsets before immunotherapy: lower PD-1⁺Tim-3^NEGCD4_EM (P = 0.005, adjusted P = 0.010), higher PD-1^NEGTim-3⁺CD8 (P = 0.002, adjusted P = 0.004), and a higher number of CTLA-4^NEG Tregs (P = 0.005, adjusted P = 0.010). We also found that an increase in Tim-3⁺ natural killer cells post- versus pre-vaccination associated with longer OS (P = 0.0074, adjusted P = 0.015). These results should be considered as hypothesis generating and should be further evaluated in larger immunotherapy trials.     

T cell profiling reveals high CD4+CTLA-4+ T cell frequency as dominant predictor for survival after Prostate GVAX/ipilimumab treatment

Immune checkpoint blockade enhances antitumor responses, but can also lead to severe immune-related adverse events (IRAE). To avoid unnecessary exposure to these potentially hazardous agents, it is important to identify biomarkers that correlate with clinical activity and can be used to select patients that will benefit from immune checkpoint blockade. To understand the consequences of CTLA-4 blockade and identify biomarkers for clinical efficacy and/or survival, an exploratory T cell monitoring study was performed in a phase I/II dose escalation/expansion trial (n = 28) of combined Prostate GVAX/ipilimumab immunotherapy. Phenotypic T cell monitoring in peripheral blood before and after Prostate GVAX/ipilimumab treatment revealed striking differences between patients who benefited from therapy and patients that did not. Treatment-induced rises in absolute lymphocyte counts, CD4⁺ T cell differentiation, and CD4⁺ and CD8⁺ T cell activation were all associated with clinical benefit. Moreover, significantly prolonged overall survival (OS) was observed for patients with high pre-treatment frequencies of CD4⁺CTLA-4⁺, CD4⁺PD-1⁺, or differentiated (i.e., non-naive) CD8⁺ T cells or low pre-treatment frequencies of differentiated CD4⁺ or regulatory T cells. Unsupervised clustering of these immune biomarkers revealed cancer-related expression of CTLA-4⁺ in CD4⁺ T cells to be a dominant predictor for survival after Prostate GVAX/ipilimumab therapy and to thus provide a putative and much-needed biomarker for patient selection prior to therapeutic CTLA4 blockade.     

Human papillomavirus type 16 E7 peptide-directed CD8+ T cells from patients with cervical cancer are cross-reactive with the coronavirus NS2 protein

Human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are required for cellular transformation and represent candidate targets for HPV-specific and major histocompatibility complex class I-restricted CD8(+)-T-cell responses in patients with cervical cancer. Recent evidence suggests that cross-reactivity represents the inherent nature of the T-cell repertoire. We identified HLA-A2 binding HPV16 E7 variant peptides from human, bacterial, or viral origin which are able to drive CD8(+)-T-cell responses directed against wild-type HPV16 E7 amino acid 11 to 19/20 (E7(11-19/20)) epitope YMLDLQPET(T) in vitro. CD8(+) T cells reacting to the HLA-A2-presented peptide from HPV16 E7(11-19(20)) recognized also the HLA-A2 binding peptide TMLDIQPED (amino acids 52 to 60) from the human coronavirus OC43 NS2 gene product. Establishment of coronavirus NS2-specific, HLA-A2-restricted CD8(+)-T-cell clones and ex vivo analysis of HPV16 E7 specific T cells obtained by HLA-A2 tetramer-guided sorting from PBL or tumor-infiltrating lymphocytes obtained from patients with cervical cancer showed that cross-reactivity with HPV16 E7(11-19(20)) and coronavirus NS2(52-60) represents a common feature of this antiviral immune response defined by cytokine production. Zero of 10 patients with carcinoma in situ neoplasia and 3 of 18 patients with cervical cancer showed > or =0.1% HPV16 E7-reactive T cells in CD8(+) peripheral blood lymphocytes. In vivo priming with HPV16 was confirmed in patients with cervical cancer or preinvasive HPV16-positive lesions using HLA-A2 tetramer complexes loaded with the E6-derived epitope KLPQLCTEL. In contrast, we could not detect E6-reactive T cells in healthy individuals. These data imply that the measurement of the HPV16 E7(11-19(20)) CD8(+)-T-cell response may reflect cross-reactivity with a common pathogen and that variant peptides may be employed to drive an effective cellular immune response against HPV.     

A DNA Vaccine Encoding a Codon-Optimized Human Papillomavirus Type 16 E6 Gene Enhances CTL Response and Anti-tumor Activity

Summary The HPV oncoproteins E6 and E7 are consistently expressed in HPV-associated cancer cells and are responsible for their malignant transformation. Therefore, HPV E6 and E7 are ideal target antigens for developing vaccines and immunotherapeutic strategies against HPV-associated neoplasms. Recently, it has been demonstrated that codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8⁺ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Our data indicated that transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8⁺ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications.     

Engineered Salmonella typhimurium expressing E7 fusion protein, derived from human papillomavirus, inhibits tumor growth in cervical tumor-bearing mice

The tumor-suppressing effects of SipB160/HPV16 E7 fusion protein, derived from human papillomavirus, and expressed in Salmonella enterica serovar typhimurium, were evaluated in a cervical cancer model. The expressed E7 protein resulted in efficacious cytotoxicity and tumor growth retardation in TC-1 cervical cancer cells. In addition, in mice bearing TC-1 tumors, live cells of Salmonella expressing HPV16 E7 were administered orally and induced immune responses through interferon-gamma and tumor necrosis factor-alpha cytokine secretion and also suppressed tumor growth (45 %) and prolonged survival (70 %) compared with the control group. These results suggested that the SipB160/HPV16 E7 fusion protein may be a candidate cancer therapeutic agent.     

Phase I trial of a recombinant yeast-CEA vaccine (GI-6207) in adults with metastatic CEA-expressing carcinoma

Yeast-CEA (GI-6207) is a therapeutic cancer vaccine genetically modified to express recombinant carcinoembryonic antigen (CEA) protein, using heat-killed yeast (Saccharomyces cerevisiae) as a vector. In preclinical studies, yeast-CEA induced a strong immune response to CEA and antitumor responses. Patients received subcutaneous vaccines every 2 weeks for 3 months and then monthly. Patients were enrolled at 3 sequential dose levels: 4, 16, and 40 yeast units (10⁷ yeast particles/unit). Eligible patients were required to have serum CEA > 5 ng/mL or > 20 % CEA⁺ tumor block, ECOG PS 0–2, and no history of autoimmunity. Restaging scans were performed at 3 months and then bimonthly. Peripheral blood was collected for the analysis of immune response (e.g., by ELISPOT assay). Twenty-five patients with metastatic CEA-expressing carcinomas were enrolled. Median patient age was 52 (range 39–81). A total of 135 vaccines were administered. The vaccine was well tolerated, and the most common adverse event was grade 1/2 injection-site reaction. Five patients had stable disease beyond 3 months (range 3.5–18 months), and each had CEA stabilization while on-study. Some patients showed evidence post-vaccination of increases in antigen-specific CD8⁺ T cells and CD4⁺ T lymphocytes and decreases in regulatory T cells. Of note, a patient with medullary thyroid cancer had substantial T cell responses and a vigorous inflammatory reaction at sites of metastatic disease. Yeast-CEA vaccination had minimal toxicity and induced some antigen-specific T cell responses and CEA stabilization in a heterogeneous, heavily pre-treated patient population. Further studies are required to determine the clinical benefit of yeast-CEA vaccination.     

Autologous cytokine-induced killer cell immunotherapy in lung cancer: a phase II clinical study

Objective

Cytokine-induced killer (CIK) cells have the ability to kill tumor in vitro and in vivo. This study was designed to evaluate the clinical efficacy of CIK cell immunotherapy following regular chemotherapy in patients with non-small cell lung cancer (NSCLC) after surgery.

Methods

A paired study, with 87 stage I–IV NSCLC patients in each group, was performed. Patients received either chemotherapy (arm 2) or chemotherapy in combination with autologous CIK cell immunotherapy (arm 1). Progression-free survival (PFS) and overall survival (OS) were evaluated.

Results

Of the 87 paired patients, 50 had early-stage disease (stage I–IIIA) and 37 had advanced-stage disease (stage IIIB–IV). Among early-stage patients, the distribution of 3-year PFS rate and median PFS time showed no statistical difference between the two groups (p = 0.259 and 0.093, respectively); however, the 3-year OS rate and median OS time in arm 1 were significantly higher than those in arm 2 (82 vs. 66 %; p = 0.049 and 73 vs. 53 months; p = 0.006, respectively). Among the advanced-stage patients, the 3-year PFS and OS rates of arm 1 were significantly higher than those of arm 2 (6 vs. 3 %; p < 0.001 and 31 vs. 3 %; p < 0.001, respectively); the median PFS and OS times in arm 1 were also significantly longer than those in arm 2 (13 vs. 6 months; p = 0.001 and 24 vs. 10 months; p < 0.001, respectively). Multivariate analyses indicated that the frequency of CIK cell immunotherapy was significantly associated with prolonged PFS (HR = 0.91; 95 % CI 0.85–0.98; p = 0.012) and OS (HR = 0.83; 95 % CI, 0.74–0.93; p = 0.001) in the arm 1.

Conclusions

The data suggested that CIK cell immunotherapy could improve the efficacy of conventional chemotherapy in NSCLC patients, and increased frequency of CIK cell treatment could further enhance the beneficial effects. A multi-center randomized trial is being carried out in our hospital to further validate these findings.     

Induction of human papillomavirus-specific CD4(+) and CD8(+) lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16- and 18-positive cervical cancer.

Human papillomavirus (HPV) type 16 (HPV 16) and HPV type 18 (HPV 18) are implicated in the induction and progression of the majority of cervical cancers. Since the E6 and E7 oncoproteins of these viruses are expressed in these lesions, such proteins might be potential tumor-specific targets for immunotherapy. In this report, we demonstrate that recombinant, full-length E7-pulsed autologous dendritic cells (DC) can elicit a specific CD8(+) cytotoxic T-lymphocyte (CTL) response against autologous tumor target cells in three patients with HPV 16- or HPV 18-positive cervical cancer. E7-specific CTL populations expressed strong cytolytic activity against autologous tumor cells, did not lyse autologous concanavalin A-treated lymphoblasts or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL), and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. Cytotoxicity against autologous tumor cells could be significantly blocked by anti-HLA class I (W6/32) and anti-CD11a/LFA-1 antibodies. Phenotypically, all CTL populations were CD3(+)/CD8(+), with variable levels of CD56 expression. CTL induced by E7-pulsed DC were also highly cytotoxic against an allogeneic HLA-A2(+) HPV 16-positive matched cell line (CaSki). In addition, we show that specific lymphoproliferative responses by autologous CD4(+) T cells can also be induced by E7-pulsed autologus DC. E7-specific CD4(+) T cells proliferated in response to E7-pulsed LCL but not unpulsed LCL, and this response could be blocked by anti-HLA class II antibody. Finally, with two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, a marked Th1-like bias (as determined by the frequency of gamma interferon- and interleukin 4-expressing cells) was observable for both CD8(+) and CD4(+) E7-specific lymphocyte populations. Taken together, these data demonstrate that full-length E7-pulsed DC can induce both E7-specific CD4(+) T-cell proliferative responses and strong CD8(+) CTL responses capable of lysing autologous naturally HPV-infected cancer cells in patients with cervical cancer. These results may have important implications for the treatment of cervical cancer patients with active or adoptive immunotherapy.     

HLA-A2-restricted peripheral blood cytolytic T lymphocyte response to HPV type 16 proteins E6 and E7 from patients with neoplastic cervical lesions

 The DNA from human papillomavirus (HPV) can be detected in 90% of cervical carcinomas. To address whether patients infected with HPV can mount efficient T cell responses to this pathogen we examined the cytotoxic T lymphocyte (CTL) response of peripheral blood mononuclear cells (PBMC) from patients with abnormal genital epithelial cells. PBMC from 11 HLA-A2⁺ patients were stimulated with CaSki, a cervical carcinoma cell line that is HPV 16⁺ and HLA-A2⁺. The CTL were screened for reactivity to the cervical carcinoma cell line C33A (HPV^–, HLA-A2⁺) transfected with the HPV 16 E6 or E7 genes or the plasmid without insert. The CTL of 1 patient showed particularly strong CaSki and HPV E6 or E7 protein-specific cytotoxicity in a HLA-A2-restricted fashion. In contrast, these CTL lysed neither a vector-only transfectant, the natural killer cell (NK) target, K562 nor the lymphokine-activated killer cell (LAK) target, Daudi. HLA-A2 restriction was demonstrated by the lack of recognition of a HLA-A2^– CaSki cell line developed in our laboratory. The CTL line was cloned and 99 clones were harvested and screened; 51 clones lysed CaSki, of which 17 did not lyse the A2^– CaSki. Of these HLA-A2^– restricted clones, 8 did not lyse C33A transfectants, 6 lysed all C33A transfectants, 3 lysed C33A-E7 only and none lysed C33A-E6 only. These data imply that, within the bulk CTL line, HLA-A2-restricted recognition of antigens was restricted to CaSki antigens, antigens common to cervical carcinoma (CaSki plus C33A), or HPV-16-E7-derived antigen on the clonal level. The E7-restricted clones were negative for recognition of known HLA-A2-binding peptides from E7. Received: 16 November 1995 / Accepted: 15 January 1996     

In vitro generated anti-tumor T lymphocytes exhibit distinct subsets mimicking in vivo antigen-experienced cells

The T-lymphocyte pool can be subdivided into naïve (Tn), effector memory (Tem), and central memory (Tcm) T cells. In this study, we characterized in vitro short-term cultured anti-tumor human T lymphocytes generated by lentiviral transduction with an anti-tumor antigen TCR vector. Within 2 weeks of in vitro culture, the cultured T cells showed a Tcm-like phenotype illustrated by a high percentage of CD62L and CD45RO cells. When the cells were sorted into populations that were CD45RO+/CD62L-(Tem), CD45RO+/CD62L+(Tcm), or CD45RO^low/CD62L+(Tn) and co-cultured with antigen-matched tumor lines, the magnitude of cytokine release from these populations for IFNγ (Tn < Tcm < Tem) and IL-2 (Tn > Tcm > Tem) mimicked the types of immune cell responses observed in vivo. In comparing cell-mediated effector function, Tn were found to be deficient (relative to Tcm and Tem) in the ability to form conjugates with tumor cells and subsequent lytic activity. Moreover, analysis of the gene expression profiles of the in vitro cultured and sorted T-cell populations also demonstrated patterns consistent with their in vivo counterparts. When Tcm and Tem were tested for the ability to survive in vivo, Tcm displayed significantly increased engraftment and persistence in NOD/SCID/γc^?/? mice. In general, a large percentage of in vitro generated anti-tumor T lymphocytes mimic a Tcm-like phenotype (based on phenotype, effector function, and increased persistence in vivo), which suggests that these Tcm-like cultured T cells may be optimal for adoptive immunotherapy.     

Intratumoral CD8+ T/FOXP3+ cell ratio is a predictive marker for survival in patients with colorectal cancer

The human immune system consists of a balance between immune surveillance against non-self antigens and tolerance of self-antigens. CD8⁺ T cells and CD4⁺ regulatory T cells (Tregs) are the main players for immune surveillance and tolerance, respectively. We examined immunohistochemically the immunological balance at the tumor site using 94 surgically resected colorectal cancer tissues. Forkhead box P3 (FOXP3)⁺ cells were considered to be Tregs in the present study. The number of intratumoral FOXP3⁺ cells (itFOXP3⁺ cells) was positively correlated with lymph node metastases (P = 0.030). itCD8⁺ T/itFOXP3⁺ cell ratio negatively correlated with pathological stages (P = 0.048). Next, relationship between the number of itCD8⁺ T cells or itFOXP3⁺ cells and survival prognosis in 94 patients who underwent a curative resection was analyzed. Only itCD8⁺ T/itFOXP3⁺ cell ratio positively correlated with disease-free survival (0.023) and overall survival (P = 0.010). Multivariate analysis indicated that itCD8⁺ T/itFOXP3⁺ cell ratio is an independent prognostic factor (P = 0.035) of overall survival. The number of itFOXP3⁺ cells positively correlated with transforming growth factor-beta TGF-β production at the tumor site (P = 0.020). In conclusion, itCD8⁺ T/itFOXP3⁺ cell ratio is a predictive marker for both disease-free survival time and overall survival time in patients with colorectal cancer. Importantly, itCD8⁺ T/itFOXP3⁺ cell ratio may be an independent prognostic factor. And, tumor-producing TGF-β may contribute to the increased number of itFOXP3⁺ cells.     

Identification of cross-clade CTL epitopes in HIV-1 clade A/E-infected individuals by using the clade B overlapping peptides

Identification of cross-clade T cell epitopes is one of key factors for the development of a widely applicable AIDS vaccine. We here investigated cross-clade CD8⁺ T cell responses between clade B and A/E viruses in chronically HIV-1 clade A/E-infected Japanese individuals. CD8⁺ T cell responses to 11-mer overlapping peptides derived from Nef, Gag, and Pol clade B consensus sequences were at a similar level to those to the same peptides found in clade B-infected individuals. Fifteen cross-clade CTL epitopes were identified from 13 regions where the frequency of responders was high in the clade A/E-infected individuals. The sequences of 6 epitopes were conserved between the clade B and clade A/E viruses whereas 9 epitopes had different amino acid sequences between the 2 viruses. CD8⁺ T cells specific for the 6 conserved epitopes recognized cells infected with the clade A/E virus, whereas those for 8 diverse epitopes recognized both the clade A/E virus-infected and clade B-infected cells. All of the cross-clade CD8⁺ T cells specific for conserved and diverse epitopes were detected in chronically HIV-1 clade A/E-infected individuals. These results show that in addition to conserved regions polymorphic ones across the clades can be targets for cross-clade CTLs.