A phase-I clinical study of autologous tumor cells plus interleukin-2-gene-transfected allogeneic fibroblasts as a vaccine in patients with cancer

Tumor cells transfected to express immunostimulatory cytokines, or admixed with similarly modified bystander cells, are able to induce immune responses against unmodified tumor cells in animal models. For treatment of human patients, a vaccine composed of autologous tumor cells and IL-2-secreting allogeneic fibroblasts was developed. Autologous tumor cells were isolated from biopsy specimens. A clone (KMST6.14) of an immortalized human fibroblast line that stably secreted 5290 IU IL-2 per 10⁶ cells and per 24 hr was obtained by cationic lipofection with an expression construct for human IL-2 and Neo^r. Fifteen patients with refractory malignant tumors received 3–4 injections of irradiated KMST6.14 and autologous tumor cells in a phase-I clinical trial. Increasing transient inflammatory responses without systemic toxicity developed at vaccination sites and after injections with irradiated tumor cells only (p < 0.05). These sites contained a dense infiltrate of CD3⁺ T cells with numbers of CD4⁺ helper cells exceeding those of CD8⁺ cytotoxic T cells (CTL). CD8⁺ T-cell lines isolated from vaccination sites of 2 malignant melanoma patients but not of renal-cell carcinoma patients exhibited a dominant lytic activity against autologous tumor cells in vitro. CD8⁺ T-cell clones established from the vaccination site of 1 of 2 renal-cell carcinoma patients preferentially lysed autologous and partially matched allogeneic renal-cell carcinoma cells. In conclusion, a vaccine composed of IL-2 gene-transfected allogeneic fibroblasts and autologous tumor cells is able to enhance specific anti-tumor T-cell responses in vivo without major side-effects. Malignant melanoma and renal-cell carcinoma appear to be promising entities for testing of similar approaches in future therapeutic trials. Int. J. Cancer, 70:269–277, 1997. © 1997 Wiley-Liss, Inc.     

Restoration of Tumor-Specific HLA Class I Restricted Cytotoxicity in Tumor Infiltrating Lymphocytes of Advanced Breast Cancer Patients by in vitro Stimulation with Tumor Antigen-Pulsed Autologous Dendritic Cells

Breast tumor infiltrating lymphocytes (TIL) are enriched in tumor-specific cytotoxic T lymphocytes (CTL), and may represent a superior source of CTL compare to peripheral blood lymphocytes (PBL), for adoptive T cell immunotherapy of breast cancer. However, the immunocompetence of TIL and the possibility to consistently restore their tumor-specific lytic activity in vitro remains an open issue. In this study we evaluated the potential of tumor antigen-pulsed fully mature dendritic cell (DC) stimulation in restoring tumor-specific cytotoxicity in anergic TIL populations from advanced breast cancer patients. In addition we have compared tumor-specific T cell responses induced by tumor antigen-loaded DC stimulation of TIL to responses induced from PBL. Although TIL were consistently non-cytotoxic after isolation or culture in the presence of interleukin-2 (IL-2), in matched experiments from three consecutive patients, tumor-lysate-pulsed DC-stimulated CD8+ T cell derived from TIL were found to be significantly more cytotoxic than PBL (p < 0.05). In addition, cytotoxicity against autologous tumor cells was more significantly inhibited by an anti-HLA class I (W6/32) MAb in TIL compared to PBL (p < 0.05). CTL populations derived from TIL and PBL did not lyse autologous EBV-transformed lymphoblastoid cell lines, and showed negligible cytotoxicity against the NK-sensitive cell line K562. Furthermore, in both CD8+ T cell populations the majority of the tumor-specific CTL exhibited a Th1 cytokine bias (IFN-^high/IL-4^low). Taken together, these data show that tumor lysate-pulsed mature DC can consistently restore tumor-specific lytic activity in non-cytotoxic breast cancer TIL. These results may have important implications for the treatment of chemotherapy resistant breast cancer with active or adoptive immunotherapy.     

Cytotoxic CD4+ and CD8+ T lymphocytes,generated by mutant p21-ras (12VAL) peptide vaccination of a patient,recognize 12VAL-dependent nested epitopes present within the vaccine peptide and kill autologous tumour cells carrying this mutation

Mutant p21-ras proteins contain sequences that distinguish them from normal ras, and represent unique epitopes for T-cell recognition of antigen-bearing tumour cells. Here, we examined the capacity of CD4⁺ and CD8⁺ T cells, generated simultaneously by mutant-ras-peptide vaccination of a pancreatic-adenocarcinoma patient, to recognize and lyse autologous tumour cells harbouring corresponding activated K-ras epitopes. The patient was vaccinated with a purified 17mer ras peptide (KLVVVGAVGVGKSALTI), containing the Gly12 → Val substitution. Responding T cells were cloned following peptide stimulation, and CD4⁺ and CD8⁺ peptide-specific cytotoxic T lymphocytes(CTL) were obtained. Transient pancreatic-adenocarcinoma cell lines(CPE) were established in cell culture from malignant ascites of the patient, and were shown to harbour the same K-ras mutation as found in the primary tumour. These cells were efficiently killed by the T-cell clones and CD8⁺-mediated cytotoxicity was HLA-class-I-restricted, as demonstrated by inhibition of lysis by anti-class-I monoclonal antibodies. By employing as targets different class-I-matched tumour cell lines expressing a 12Val mutation, we were able to demonstrate HLA-B35 as the restriction molecule, and further use of peptide-sensitized EBV-B cells as target cells identified VVVGAVGVG as the nonamer peptide responsible for CD8⁺-T-cell recognition. These data demonstrate that peptide vaccination with a single mutant p21-ras-derived peptide induces CD4⁺ and CD8⁺ CTL specific for nested epitopes, including the Gly → Val substitution at codon 12, and that both these T-cell sub-sets specifically recognize tumour cells harbouring the corresponding K-ras mutation. Int. J. Cancer 72:784–790, 1997. © 1997 Wiley-Liss, Inc.     

Histocompatibility Leukocyte Antigen-A2-restricted and Tumor-specific Cytotoxic T Lymphocytes from Tumor-infiltrating Lymphocytes of a Patient with Testicular Embryonal Cancer

T lymphocytes play an important role in tumor rejection. To understand T cell-mediated specific immunity at the tumor site of testicular embryonal cancer, we investigated whether interleukin-2 (IL-2)-activated tumor-infiltrating lymphocytes (TIL) of a patient with testicular embryonal cancer show histocompatibility leukocyte antigen (HLA)-class I-restricted and tumor-specific cytotoxicity. We established a CD3⁺CD4^-CD8⁺ cytotoxic T lymphocyte (CTL) line from the IL-2-activated TIL of a 37-year-old patient with testicular embryonal cancer. A 6 h ⁵¹Cr-release assay was performed to measure the cytotoxicity of the CTL. The CD3⁺CD4^-CD8⁺ CTL line showed cytotoxicity against HLA-A2⁺ tumor cells, including freshly isolated autologous tumor cells, adenocarcinoma cell lines from various organs (lung, breast, pancreas, colon and kidney) and squamous cell carcinomas (esophagus and oral cavity). No other cell lines examined, including an autologous tumor cell line and HLA-A2" tumor cell lines, were lysed by this CTL line. These results suggest the existence of HLA-A2-restricted and tumor-specific CTL at the tumor site of testicular embryonal cancer.     

Usage of T-cell receptor Vβ chain genes in fresh and cultured tumor-infiltrating lymphocytes from human melanoma

Tumor-infiltrating lymphocytes (TIL) freshly obtained from human malignant melanomas as well as the same TIL grown in the presence of interleukin 2 (IL2) were studied for gene expression of the T-cell receptor (TCR) variable β regions (Vβ). To perform the TCR-Vβ analysis, total RNA was isolated from TIL and reverse-transcribed into cDNA, which was then amplified by PCR using 22 different 5′ primers specifically recognizing the sequences of 20 Vβ gene families and a 3′ primer annealing to the constant region of the β chain. The TCR-α constant region (Cα) gene was co-amplified as a standard for the calculation of the percentage of each TCR-Vβ gene expressed. The frequency of individual Vβ regions expressed on TIL was computed from the ratio of cpm Vβ to cpm Ca for each Vβ region in relation to the total of all 22 ratios. With fresh TIL obtained from 8 different melanomas, oligoclonal distribution of Vβ genes expressed on TIL was observed, in comparison with a broader and unrestricted distribution seen with peripheral-blood T cells of 8 normal individuals. The oligoclonal patterns of Vβ-gene expression in fresh melanoma TIL were distinct in every tumor. Several of the Vβ-genes usually expressed in normal PBL were not expressed in fresh TIL. In melanoma TIL cultured in the presence of IL2 and IL4 and in the absence of autologous tumor (AuTu) or antigen-presenting cells for 23 to 65 days, selection of T-cell lines expressing a restricted number of Vβ genes occurred. Although in 4/5 TIL cultures this selection involved the Vβ7 gene, no relationship could be established between Vβ gene expression in fresh TIL and that in T-cell lines outgrowing in long-term cultures. Selection in culture of CD3⁺ CD8⁺ T-cell lines with Vβ-gene expression restricted to I or 2 Vβ families did not correlate with the presence or level of AuTu cytotoxicity mediated by these T cells. The results indicate that in TIL cultures random selection of T-cell lines with reactivity not relevant to AuTu may account for poor expression or loss of AuTu cytotoxicity by most TIL cultured long-term in the presence of cytokines and in the absence of specific antigenic stimulation.     

Isolation and characterization of tumor-infiltrating lymphocytes from cervical carcinoma

The evidence that virus-induced tumors generally elicit T-cell responses prompts the notion that HPV-related cervical carcinoma would be amenable to treatment by T-cell-mediated adoptive therapy. Therefore, we cultured and cloned tumorinfiltrating lymphocytes (TIL) from a patient with cervical carcinoma and studied the in vitro characteristics of these TIL by using the established autologous tumor-cell line. After stimulation of bulk TIL cultures with 1,000 Units/ml recombinant interleukin 2 (rIL-2), followed by limiting dilution, T-cell clones were generated in the presence of 20 U/ml rIL-2 and irradiated autologous tumor cells, PBLs and EBV-transformed B-cell lines. Phenotypically, all clones were CD3/CD8-positive with a heterogeneous CD56 expression. All expressed preferential cytolytic activity against autologous tumor cells, did not lyse autologous lymphoblasts, and were cytotoxic against the NK-sensitive cell line K562. A minor lytic capacity was detectable on allogeneic cervical tumor-cell lines or tumor-cell lines of other histologic types. Cytotoxicity against the autologous tumor could be inhibited by anti-CD3, anti-CD8 and anti-ICAMI but not by anti-HLA class-1 (W6/32, B9.12.I), anti-allele-specific HLA determinants and anti-LFA-3 antibodies. We demonstrate a highly specific autologous lytic activity of cervical carcinoma TIL, in which a CD3-associated surface antigen recognition is involved. These results may prove useful in further studies on adoptive immunotherapy of cervical cancer patients. © 1994 Wiley-Liss, Inc.     

Clonal analysis of tumor-infiltrating lymphocytes from human primary and metastatic liver tumors

Phenotypic and functional characteristics of tumor-infiltrating lymphocytes (TIL) obtained from human primary and metastatic liver tumors were studied. Lymphocytes isolated from 18 tumors and autologous (A) peripheral blood (6 cases) were phenotyped by 2-color flow cytometry and cloned in a limiting dilution system, which allows virtually all normal T lymphocytes to proliferate; 70-80% of fresh TIL were T cells (i.e., CD3+), and the ratio of CD4+/CD8+ cells was 1.2 in both primary and metastatic liver tumors. TIL contained significantly more CD56+ (NKHI+) cells, half of which were CD3+CD56+, CD3+CD25+ cells and CD3+HLA-DR+ cells, than A-PBL. The frequencies of proliferating T-cell precursors (PTL-p) and cytolytic T-lymphocyte precursors (CTL-p) reactive with K562, allogeneic tumor cells and autologous tumor cells, were determined. Mean PTL-p frequencies for TIL from hepatocellular carcinomas, cholangiocarcinomas and metastatic liver tumors were 0.52 (0.22-0.83), 0.10 (0.05-0.16) and 0.16 (0.01-0.30), respectively. The frequency of CTL-p with natural-killer-like activity was lower in TIL than in A-PBL. The frequency of CTL-p for autologous tumor cells in fresh TIL isolated from primary liver tumors was 0.02-0.13 and 12/81 clones were reactive against autologous tumor. In contrast, only 1/66 TIL clones obtained from colon carcinomas metastatic to liver showed autotumor reactivity. No clones reactive with autologous tumor were obtained from peripheral blood of patients with liver cancer. These data indicate that substantial differences in anti-tumor functions of TIL between primary and metastatic liver tumors exist, which can be detected at a clonal level.     

GITR ligation enhances functionality of tumor-infiltrating T cells in hepatocellular carcinoma

No curative treatment options are available for advanced hepatocellular carcinoma (HCC). Anti-PD1 antibody therapy can induce tumor regression in 20% of advanced HCC patients, demonstrating that co-inhibitory immune checkpoint blockade has therapeutic potential for this type of cancer. However, whether agonistic targeting of co-stimulatory receptors might be able to stimulate anti-tumor immunity in HCC is as yet unknown. We investigated whether agonistic targeting of the co-stimulatory receptor GITR could reinvigorate ex vivo functional responses of tumor-infiltrating lymphocytes (TIL) freshly isolated from resected tumors of HCC patients. In addition, we compared GITR expression between TIL and paired samples of leukocytes isolated from blood and tumor-free liver tissues, and studied the effects of combined GITR and PD1 targeting on ex vivo TIL responses. In all three tissue compartments, CD4⁺FoxP3⁺ regulatory T cells (Treg) showed higher GITR⁻expression than effector T-cell subsets. The highest expression of GITR was found on CD4⁺FoxP3^hiCD45RA⁻ activated Treg in tumors. Recombinant GITR-ligand as well as a humanized agonistic anti-GITR antibody enhanced ex vivo proliferative responses of CD4⁺ and CD8⁺ TIL to tumor antigens presented by mRNA-transfected autologous B-cell blasts, and also reinforced proliferation, IFN-γ secretion and granzyme B production in stimulations of TIL with CD3/CD28 antibodies. Combining GITR ligation with anti-PD1 antibody nivolumab further enhanced tumor antigen-specific responses of TIL in some, but not all, HCC patients, compared to either single treatment. In conclusion, agonistic targeting of GITR can enhance functionality of HCC TIL, and may therefore be a promising strategy for single or combinatorial immunotherapy in HCC.     

Generation of CD4+ cytotoxic T lymphocytes stimulated by immobilized anti-CD3 monoclonal antibody and interleukin-2 in cancer patients

The proliferation of autologous tumor-reactive cytotoxic T lymphocytes (CTL), induced by autologous mixed lymphocyte tumor-cell culture, was remarkably enhanced by activation with immobilized anti-CD3 monoclonal antibody (MAb) and interleukin-2 (IL-2), as compared with IL-2 alone. The activated CTL exhibited high cytotoxicity against autologous tumor cells. Cytotoxicity against autologous tumor cells was inhibited by anti-HLA-DR MAb. In negative selection with immunomagnetic beads, cytotoxicity against autologous tumor cells was inhibited by the elimination of CD4⁺ cells. The major cell-surface antigens of the activated CTL were CD3⁺, CD4⁺, CD25⁺, CD45RO⁺and CD45RA^-, suggesting helper T cells, and the activated CTL produced IL-2. It is concluded that the CTL activated by immobilized anti-CD3 MAb and IL-2 were CD4 cells that had both killer and helper functions. Our findings indicate that adoptive immunotherapy using these activated CTL would be effective in cancer patients. © 1995 Wiley-Liss, Inc.     

Tumor‐reactive CD4+CD8αβ+ CD103+ αβT cells: A prevalent tumor‐reactive T‐cell subset in metastatic colorectal cancers

High level of T‐cell infiltration in colorectal carcinomas (CRCs) is a good prognostic indicator, but the tumor reactivity of this infiltrate (tumor infiltrating lymphocytes [TIL]) is poorly documented. This study examined the presence, phenotype and functional features of tumor‐reactive lymphocytes in human CRC. Freshly dissociated TIL and T cell lines were isolated from CRC samples and from some paired normal colonic mucosa. Four tumor cell lines were obtained. Autologous tumor reactivity of CRC TIL and tumor‐reactive cell features were analyzed. We demonstrate the presence among CRC TIL of variable fractions (up to 18%) of double positive CD4⁺CD8αβ⁺ (DP) αβ T cells. Interestingly, a high proportion (16–20%) of this TIL subset displayed tumor reactivity, whilst this was the case for no or few single positive TIL. Low levels of DP TIL were found in most CRC samples and in normal colonic mucosa, but these cells were higher in metastatic CRC. Furthermore, we showed that DP TIL were polyclonal, restricted by HLA class‐I, proliferated poorly and secreted higher amounts of IL‐4 and IL‐13 than single positive T cells, on cognate or CD3 stimulation. DP CRC TIL also expressed CD103, confirming their mucosal origin. Increased frequencies of tumor‐reactive DP TIL in metastatic CRC suggest that these cells play a role in the metastatic process of this cancer. Based on their high secretion of IL‐4 and IL‐13 and on previously described roles of these cytokines in cancers, we postulate that DP TIL could favor CRC growth or metastasis and/or downmodulate immune responses to these tumors.     

Differences in the recognition of tumor-specific CD8+ T cells derived from solid tumor,metastatic lymph nodes and ascites in patients with gastric cancer

We established gastric cancer-specific CD8⁺ T-cell (T_CD8⁺) lines derived from different lymphocyte sources in the same patients by repeated stimulation with mitomycin-C-treated autologous tumor cells with low-dose interleukin-2, and we compared recognition patterns among the T_CD8⁺ derived from solid tumor, lymph node metastasis and ascites in the same patient (n = 3) to determine their similarities and differences for therapeutic purposes. We confirmed that gastric cancer-specific T_CD8⁺ lines can be isolated, in a MHC class I-restricted manner, from solid tumors, metastatic lymph nodes and malignant ascites. T_CD8⁺ lines derived from tumor-infiltrating lymphocytes (TIL) in solid tumor recognized autologous tumor cells derived from solid tumor, but not autologous tumor cells derived from ascites or metastatic lymph node, while T_CD8⁺ lines derived from tumor-associated lymphocytes (TAL) in malignant ascites recognized autologous tumor cells derived from ascites, but not tumor cells from solid tumor or metastatic lymph node. Furthermore, T_CD8⁺ lines derived from regional lymph node lymphocytes (RLNL) recognized autologous tumor cells derived from metastatic lymph nodes, but not tumor cells derived from ascites. No significant differences were seen in MHC class I expression among the tumors derived from solid tumor, lymph node metastasis or ascites in the same patient. This suggests that there are differences of recognition patterns among the TILs, TALs and RLNLs in the same patient and that it is important to consider the source of lymphocytes, e.g., a combination of TILs, TALs and RLNLs, for adoptive immunotherapy in gastric cancer patients. Int. J. Cancer 71: 978-981, 1997. © 1997 Wiley-Liss Inc.     

Precursor frequency analysis of human cytolytic T lymphocytes directed against autologous melanoma cells.

Limiting numbers of peripheral-blood mononuclear cells (PBMC) from melanoma patients were stimulated with irradiated autologous tumor cells in the presence of interleukins-2 and -4 and in the absence of feeder cells. The responder cells were restimulated every week. After 2 to 4 weeks, the microcultures were tested for their lytic activity against the autologous tumor cells. Significant lysis of the tumor cells was observed with a fraction of these microcultures, whereas no lysis was observed with control microcultures seeded without stimulator melanoma cells. Because our aim was to measure the precursor frequency of CTL showing specificity for the tumor, and not that of NK-like effectors that were also capable of lysing the melanoma cells, we used cold-target inhibition with an excess of NK target K562 to inhibit the NK-like activity. Microcultures whose lysis on the tumor cells was not abolished by K562 competition were observed. The specificity of these CTL clones was confirmed by the absence of lytic activity on autologous T-cell blasts. The numbers of microcultures with anti-tumor CTL activity fitted the zero-order of the Poisson distribution equation, indicating that they resulted from the activity of single T-cell clones. The frequency of anti-tumor CTL precursor cells (CTL-P) of 7 melanoma patients ranged from 1/900 to 1/33,000. Frequencies of anti-tumoral CTL-P were higher and NK-like effectors were less frequent when sorted CD8+ T lymphocytes were used as responder cells.     

Diverse effect of cytokine treatment of tumor cells on specific versus non-specific cytotoxicity

(First submitted 15 Nov 1991;accepted 11 December 1991) The effect of IFN-γ and TNF-α treatment of an ovarian carcinoma line on the sensitivity to lysis by specific CTL clones and non-specific Tumor Associated Lymphocytes (TAL), isolated from the ascites fluid, was analyzed. Thein vitro established TAL line displayed a non-specific lytic activity against the autologous tumor as well as against several allogeneic tumor lines. Pretreatment with IFN-γ alone, or in combination with TNF-α, rendered the carcinoma line less susceptible to lysis by the autologous TAL line. Conversely, susceptibility to lysis by tumor specific T cell clones, isolated from the TAL line, was increased as a result of cytokine pretreatment. Several TCR-α/β⁺, CD8⁺ T-cell clones showing a more specific pattern of lysis against the autologous tumor were isolated. Lysis of the autologous tumor by these clones involved the TCR-α/β via a MHC-class I restricted mechanism dependent on the adhesion molecules ICAM-1 and LFA-3, as inferred from antibody blocking studies. The enhanced sensitivity to specific CTL clones seen after cytokine treatment may be related to theenhanced expression of ICAM-1 molecules on the ovarian carcinoma. These results have implications for cytokine based immunotherapy, where IFN-γ may enhance the effects of tumor associated specific CTL while decreasing that of non-specific effector cells.     

Interleukin-12 Augments the Generation of Autologous Tumor-reactive CD8+ Cytotoxic T Lymphocytes from Tumor-infiltrating Lymphocytes

Human tumor-infiltrating lymphocytes (TIL) were obtained from breast cancer, renal cancer or neuroblastoma to investigate the generation of autologous tumor-reactive CD8⁺ cytotoxic T lymphocytes (CTL). When TIL were cultured with interleukin (IL)-2 (100 U/ml), the growth of TIL peaked around 8–10 days after the initiation of culture. In contrast, the proliferation of TIL cultured with IL-2 plus IL-12 peaked around 4–5 days after culture and tumor cells rapidly disappeared from the culture. To determine the generation of autologous tumor-reactive CD8⁺ CTL, TIL-derived CD8⁺ T cells were separated by FACStar. Both IL-2-activated and IL-2 plus IL-12-activated TIL-CD8⁺ T cells showed the same level of lymphokine-activated killer activity against a variety of tumor cells. However, TIL-CD8⁺ T cells activated with IL-2 plus IL-12 revealed greatly augmented cytotoxicity against autologous tumor cells compared with that induced by IL-2 alone. The autologous tumor cell-killing activity of TIL-CD8⁺ CTL was significantly inhibited by the addition of F(ab)₂ anti-CD3 monoclonal antibody, indicating that these CTL recognize autologous tumor antigen through T cell receptor. These results imply that IL-12 is a novel cytokine which facilitates the generation of autologous tumor-reactive CD8⁺ CTL from TIL.     

Adoptive transfer of ex vivo-activated memory T-cell subsets with cyclophosphamide provides effective tumor-specific chemoimmunotherapy of advanced metastatic murine melanoma and carcinoma

Autolymphocyte therapy (ALT) is adoptive cellular therapy of neoplastic disease using ex vivo activation of autologous (human) or syngeneic (murine) lymphocytes from tumor-bearing hosts (TBH) by low doses of anti-CD3 monoclonal antibody (MAb) and a mixture of previously prepared autologous cytokines (T3CS). Ex vivo activation by T3CS without tumor antigen results in expansion of CD44⁺ (memory) T cells. These memory T cells (ALT cells) mediate in vivo anti-tumor specificity and with cyclophosphamide (CY) are capable of curing metastatic disease in murine TBH. To determine whether CY could enhance the effectiveness of CD4⁺ or CD8⁺ subsets of ALT cells, C57BL/6J TBH with B16 melanoma or Lewis lung (3LL) carcinoma were treated with adoptive chemoimmunotherapy (ACIT) using CD4-depleted or CD8-depleted ALT cells and CY. ALT cells were derived from splenocytes of B16 or 3LL-TBH and activated ex vivo with T3CS. Depletion of CD4⁺ or CD8⁺ T cells was performed before or after activation with T3CS. B16-TBH or 3LL-TBH that received ACIT using CY with B16-derived or 3LL-derived CD8-depleted ALT cells, respectively, demonstrated cure of metastatic disease regardless of whether CD8⁺ T cells were depleted before or after T3CS activation. B16 or 3LL-TBH that received ACIT using CY with B16 or 3LL-derived CD4-depleted ALT cells also cured metastatic disease but only if CD4⁺ T cells were depleted after T3CS activation. Interleukin (IL)-2 added to pre-T3CS CD4-depleted ALT cells cultured with T3CS restored anti-tumor activity when combined with CY. TBH cured by ACIT using CY and ALT-cell subsets derived from syngeneic TBH with the identical tumor displayed tumor-specific immunity in rejecting a lethal challenge of identical but not reciprocal tumor. TBH given ACIT using CY and ALT-cell subsets derived from splenocytes of syngeneic TBH with reciprocal tumors rejected lethal challenges of both tumors. Tumor specificity measured by interferon (IFN)-γ and ⁵¹Cr-release assays was demonstrated in pre- or post-T3CS/CD8-depleted, post-T3CS/CD4-depleted and pre-T3CS + IL-2/CD4-depleted ALT-cell subsets. Our data demonstrate that ACIT using CY combined with ex vivo T3CS-activated CD44⁺ memory T-cell subsets conveys long-term tumor-specific immunity. © 1995 Wiley-Liss, Inc.     

Intratumoral T cell subset ratios and Fas ligand expression on brain tumor endothelium

Summary Introduction: T cell presence in TIL, and the ratio of CD8⁺ and CD4⁺ T cell subsets in particular, can correlate with tumor prognosis in some tumors, although the significance of such infiltration into glioma is controversial. However, gliomas represent a lower extreme in their extent of T cell infiltration, and are thus useful in assessing factors that can decrease T cell presence within tumor tissue. Fas ligand, a pro-apoptotic cell surface protein, may play a key role in reduction of T cells in tumor tissue. Objective: To assess the level of FasL expression on brain tumor endothelium and to correlate this with relative levels of CD4⁺ and CD8⁺ T cell subsets in TIL from brain tumors. Methods: CD3⁺, CD4⁺, and CD8⁺ cells were quantified in fresh TIL by flow cytometry. Paraffin embedded sections of tumors, including meningiomas and gliomas as well as extracranial malignancies, underwent immunohistochemical staining for FasL and Von-Willebrand’s factor (Factor VIII) to determine expression levels of endothelial FasL. Results: FasL expression was high in aggressive intracranial malignancies compared to more indolent neoplasms, and correlated inversely with CD8⁺/CD4⁺ TIL ratios in all tumor classes combined (ANOVA,p<0.05). Conclusion: Low levels of T cells within TIL, as well as low CD8⁺/CD4⁺ TIL ratios appear to be a property of parenchymal tumor presence. Together with the inverse correlation seen between FasL expression and CD8⁺/CD4⁺ TIL ratios, the high levels of endothelial FasL expression in gliomas suggests that FasL decreases T cell presence in brain tumors in a subset-selective manner, thus contributing to glioma immune privilege.     

Human normal CTL clones: Generation and properties

This study reports the production of clones of human killer T cells grown in the presence of TCGF⁴ following sensitization in MLC against (1) allogeneic cells, (2) autologous B^ebv+ lymphocytes, or (3) autologous lymphoma cells. Sensitization against the tumor cells required the addition of macrophages. The expression of the cytotoxic activity of the cloned T lymphocytes required re-stimulation with the specific stimulator cells. The cytotoxic activity seemed to be MHC-restricted, since (1) cloned allosensitized CTL lysed their corresponding allogeneic targets, but did not lyse autologous B^ebv+ cells or K562 cells; (2) cloned CTL sensitized against autologous B^ebv+ cells lysed their autologous targets but not allogeneic B^ebv+ targets or K562 cells; and (3) cloned CTL sensitized against autologous Burkitt lymphoma cells lysed their corresponding lymphoma targets or autologous B^ebv+ targets but did not lyse allogeneic lymphoma cells or B^ebv+ cells from the same allogeneic donors. The cloned CTL were homogeneous in expressing the OK T8 molecules and in being negative for T4, T10 or M1. At any given time, 25-45% of the cloned cells manifested lytic activity. The ultrastructural properties and cell surface OK T markers were different from those of cloned human NK cells. Emphasis is focused on the differences between the structural, functional and culture characteristics of CTL clones produced by direct isolation of MLC responder cells forming conjugates with specific target cells and those of clones from transformed T-cell lines or from T hybridomas.