Autolymphocyte therapy. III. Effective adjuvant adoptive cellular therapy with in vivo anti-tumor specificity against murine melanoma and carcinoma using ex-vivo-activated memory T-lymphocytes.

Autolymphocyte therapy (ALT) is adoptive cellular therapy of neoplastic disease based upon ex vivo activation of lymphocytes by either the supernatant derived from a previously prepared one-way mixed lymphocyte culture (MLC) or using low doses of the mitogenic monoclonal antibody OKT3 and a mixture of previously prepared cytokines (T3CS). We have previously demonstrated that nonspecific ex vivo activation of splenocytes from murine tumor-bearing hosts (TBH) using an MLC-supernatant or T3CS without the use of tumor antigen results in the expansion of the CD44+ (memory) T-cell subset. These CD44+ T-cells are the principal mediators of anti-tumor specificity in the ALT-cell population in advanced metastatic murine tumors and are able to protect against tumor challenge in healthy syngeneic mice (HSM). To determine if ALT is effective in an adjuvant setting, C57BL/6J splenocytes from HSM and TBH with B16 melanoma or Lewis lung (3LL) carcinoma were activated ex vivo using T3CS. Mice were implanted with either B16 melanoma or 3LL carcinoma and then underwent surgical excision of tumor. Tumor-excised mice (TEM) then received small numbers (10(6)) of ALT-cells derived from 3LL-TBH or B16-TBH splenocytes, HSM-derived ALT-cells, fresh splenocytes derived from 3LL-TBH or B16-TBH, or CD44-depleted ALT-cells. Significant anti-tumor activity as shown by prolonged survival (Day 100), cure of disease, and rejection of a local and systemic tumor rechallenge was demonstrated in 3LL-TEM that received B16-derived ALT-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ex vivo activated memory T-lymphocytes as adoptive cellular therapy of human renal cell tumour targets with potentiation by cis-diamminedichloroplatinum(II).

OBJECTIVE: To determine if cis-diamminedichloroplatinum(II) (CDDP) enhances, by immunomodulation, ex vivo anti-tumour cytotoxicity of autolymphocyte therapy (ALT) against a chemotherapy-resistant tumour, and if lysis is mediated through T-cells, NK-cells, or both. MATERIALS AND METHODS: Human renal cell carcinoma (RCC) target cells were derived from surgical specimens and incubated in complete medium (CM) with CDDP, or in CM alone (control group). ALT-cells were prepared from autologous whole peripheral blood mononuclear cells (PBMC) or NK-cell (CD56)-depleted PBMC obtained before surgery. Tumour cells from each group were labelled with chromium-51(51Cr) and used as targets for ALT-cells and PBMC in a standard (4 h) and delayed (18 h) 51Cr-release assay at varying effector/target ratios (E:T). RESULTS: Tumour cells incubated in CDDP showed enhanced lysis, as measured by the 51Cr-release assay, at all E:T tested. This lysis was significantly greater during the 18 h assay and when ALT-cells were used as the effector cells rather than PBMC. Depletion of CD45RO+ (memory) T-cells from the ALT cell population precluded both the 4 and 18 h tumour cell lysis. Depletion of NK-cells (CD56+) diminished the ex vivo lysis of autologous targets during the 4 but not the 18 h assay. ALT-cells derived from two patients demonstrated ex vivo tumour-specificity against autologous and allogeneic RCC. CONCLUSIONS: These data suggest that: (i) ex vivo activated memory T-cells are the principal component demonstrating significant tumour-specific cytotoxicity of ALT-cells against RCC tumour targets; (ii) CDDP may alter the physical properties of tumour cells rendering them susceptible to immune-mediated attack; (iii) the combination of ALT and CDDP may lead to increased therapeutic efficacy in patients with metastatic RCC.

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)-gamma 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.

Ex vivo activated memory T-lymphocytes as adoptive cellular therapy of human soft-tissue sarcoma targets with potentiation by cis-diamminedichloroplatinum(II).

Autolymphocyte therapy (ALT) is tumor-specific, adoptive cellular therapy of neoplastic disease using nonspecific ex vivo activation of autologous peripheral blood lymphocytes (PBL), which are composed primarily of memory T-cells (ALT-cells) and are active in patients with metastatic renal cell carcinoma and melanoma. Ex vivo pretreatment of tumor target cells with certain chemotherapeutic agents can enhance susceptibility to lysis by antitumor lymphocytes. To determine if cis-diamminedichloroplatinum(II) (CDDP) enhances ex vivo antitumor cytotoxicity of ALT-cells and if this lysis is mediated by T- and/or NK-cells and is human leukocyte antigen (HLA)-restricted, human soft tissue sarcoma (STS) target cells were derived from primary and metastatic surgical specimens and were incubated with and without CDDP. ALT-cells were prepared from autologous PBL obtained prior to surgery. Primary (PSTS) and metastatic (MSTS) target cells from each group were labelled with chromium 51 (51Cr) and used as targets for ALT-cells, CD45-depleted ALT-cells, CD56 (NK)-depleted ALT-cells, and PBL in a standard (4-hour) and delayed (18-hour) 51Cr release assay. Interferon-gamma (IFN-gamma) release was measured as an indication of antitumor effect and recognition by the noncytolytic lymphocytes in ALT-cells. Primary tumor target cells incubated in CDDP showed enhanced lysis as measured by the 51Cr release assay compared to non-CDDP-treated controls. Metastatic tumor target cells showed less lysis than the primary targets, although this was enhanced by pretreating metastatic tumor targets with CDDP. Lysis of all tumor targets was significantly greater when ALT-cells were used as the effector cells rather than PBL. Depletion of memory T-cells abrogated ex vivo lysis. Depletion of NK cells (CD56+) affected ex vivo lysis of autologous targets during the 4-hour but not the 18-hour assay. Ex vivo ALT-cell lysis and IFN-gamma release against only the autologous tumor targets confirmed tumor-specificity in one patient. Restriction of ALT-cell lysis and IFN-gamma release against HLA-A2+ autologous and one allogeneic HLA-A2+ STS tumor target, but not other non-STS targets, was demonstrated in another patient. These data suggest that CDDP may help render STS susceptible to tumor-specific, immune-mediated attack and that the combination of ALT and CDDP may lead to effective tumor-specific chemoimmunotherapy in patients with metastatic STS.

Newspaper reporting of the medical literature.

OBJECTIVE: To examine whether the media are providing information to the public about important medical advances in a timely manner and whether the degree of importance is associated with other aspects of newspaper reporting (presence, extent, and prominence). DESIGN: The authors explored the amount, extent, prominence, and timeliness of newspaper coverage received by New England Journal of Medicine and JAMA articles published in 1988, by searching ten leading U.S. newspapers. The journal articles were independently rated based on the public's need to know the medical information contained in the article. The intraclass reliability coefficient for this need-to-know importance score was 0.77. MEASUREMENTS AND MAIN RESULTS: Overall, 35% of the journal articles received newspaper coverage (276/786). The articles were frequently covered by more than one newspaper [extensive coverage (161/276, 58%)] and often appeared on the front page [prominent coverage (42/276, 15%)]. Articles considered most important to the public (92/786, 12%) received more extensive and prominent coverage than did less important articles (p < 0.01). More than three fourths of the newspaper stories appeared within two days of the journal article's issue date. Stories about the most important articles appeared sooner than did those about the less important articles (p < 0.0001). CONCLUSIONS: Articles reported in two prominent medical journals are often viewed as being important to the public, and these articles are receiving newspaper coverage that is extensive, prominent, and timely. This is particularly true for those articles considered most important to the public.

Adoptive transfer of ex vivo activated memory T-cells with or without cyclophosphamide for advanced metastatic melanoma: results in 36 patients.

Autolymphocyte therapy (ALT) is the infusion of autologous peripheral blood mononuclear cells (PBMC) activated ex vivo by a cytokine-rich supernatant (T3CS) generated from a previous autologous lymphocyte culture using low doses of the anti-CD3 mitogenic monoclonal antibody. The mechanism of action is enhancement of a recall response by CD45RO+ (memory) T-cells (ALT cells) to host tumour without dependence on exogenous interleukin (IL)-2. The existence of anti-tumour-specific T-cells in melanoma patients has been well described, and efforts to utilise them therapeutically have achieved modest tumour response rates. However, few long-term survival data have been reported. From 1986 to 1992, we treated 36 patients with disseminated melanoma using ALT alone (26 patients) or adoptive chemoimmunotherapy using ALT and cyclophosphamide (CY) (10 patients). Over this time period, the cell activation method evolved from using cytokine supernatants derived from a one-way allogeneic mixed lymphocyte culture (MLCS), to the current practice of utilising anti-CD3 and autologous cytokines (T3CS). There were 21 men and 15 women, average age 57 years, range 30-82. 27 had failed prior therapies and 9 had no prior therapy. A total of 161 infusion of ALT cells were given: 65 with cells activated in MLCS and 96 with T3CS. There were no grade 3 adverse events, and an approximate 20% incidence of grades 1 and 2 reactions to ALT-cell infusions. Transient cytopenias were seen in patients receiving CY. Sixty-one per cent (22/36) of patients received the planned six ALT-cell infusions, while 39% did not due to progressive disease. In 33 evaluable patients, there were four complete responses, four partial responses and 6 patients with stable disease (SD). Responding patients and those with SD had prolonged survival compared to historical controls when matched for number of organ systems involved. Ex vivo depletion of CD45RO+ T-cells revealed preferential lysis of autologous and HLA-A-matched melanoma targets that was dependent on these memory T-cells. These data suggest that adoptive cellular therapy using ex vivo activated memory T-cells with and without CY is active, has low toxicity, is tumour-specific and can result in clinical benefit in patients with disseminated melanoma.

Adoptively transferred ex vivo activated memory T cells with cyclophosphamide: effective tumor-specific chemoimmunotherapy of advanced metastatic murine melanoma and carcinoma.

Autolymphocyte therapy (ALT) is adoptive cellular therapy of neoplastic disease based upon ex vivo activation of autologous peripheral blood mononuclear cells by either the supernatant derived from a previously prepared one-way mixed lymphocyte culture (MLC) or using low doses of the mitogenic monoclonal antibody anti-CD3 and a mixture of previously prepared autologous cytokines (T3CS). We have previously demonstrated that nonspecific ex vivo activation of splenocytes from murine tumor-bearing hosts (TBH) using an MLC supernatant or T3CS without the use of tumor antigen results in the expansion of the CD44+ (memory) T-cell subset. These memory T cells or autolymphocytes (ALT cells) mediate in vivo specificity when infused into murine TBH. To determine if cyclophosphamide (CY) could enhance the effectiveness of these ALT cells, C57BL/6J TBH with B16 melanoma or Lewis lung (3LL) carcinoma were treated with adoptive chemoimmunotherapy (ACIT) consisting of ALT cells and varying doses of CY. ALT cells were derived from the splenocytes of C57BL/6J TBH with B16 melanoma or 3LL carcinoma and activated ex vivo with T3CS. 3LL or B16 TBH received ALT cells derived from 3LL TBH splenocytes, B16 TBH splenocytes, CY alone, or ACIT with ALT cells and CY. Significantly greater antitumor activity as demonstrated by a decreased number of lung metastases (Day 21) and cure of primary and metastatic disease (Day 100) was seen in B16 or 3LL TBH that received ACIT using CY with B16-derived and 3LL-derived ALT cells, respectively, rather than ALT cells alone, CY alone, or ACIT with CY and reciprocal tumor-derived ALT cells. TBH cured by ACIT using CY and ALT cells derived from syngeneic TBH with the identical tumor displayed tumor-specific immunity as they were able to reject a lethal challenge of their previous B16 or 3LL tumor but not reciprocal tumors. TBH cured by ACIT using CY and ALT cells derived from splenocytes of syngeneic TBH with reciprocal tumors were able to reject lethal challenges of both tumors. These data demonstrate that effective ACIT using CY combined with ex vivo T3CS-activated CD44+ memory T cells from TBH conveys long-term tumor-specific immunity.

Autolymphocyte therapy. II. Dependence of in vivo anti-tumor specificity and long-term immunity against murine melanoma and carcinoma on ex vivo activated donor memory T-cells.

Autolymphocyte therapy (ALT) is tumor-specific adoptive cellular therapy of neoplastic disease in human tumor-bearing hosts based upon nonspecific ex vivo activation of autologous peripheral blood lymphocytes. We have previously demonstrated that nonspecific ex vivo activation of splenocytes from murine tumor-bearing hosts using a mixed-lymphocyte culture supernatant without tumor antigen results in the expansion of the CD44+ (memory) T-cell subset and that depletion of these CD44+ T-cells results in the abrogation of all in vivo anti-tumor effects. To examine other means of generating anti-tumor-specific effectors, splenocytes of C57BL/6J healthy syngeneic mice and mice with B16 melanoma (B16 mice) or Lewis lung (3LL) carcinoma (3LL mice) were activated ex vivo using low doses of the mitogenic monoclonal antibody OKT3 and a mixture of previously prepared autologous cytokines (T3CS). Immunophenotypic studies of splenocytes pre- and postactivation by T3CS revealed preferential expansion of Thy-1.2+CD44+ (memory) T-cells. 3LL and B16 mice received ALT cells derived from splenocytes of 3LL mice, B16 mice, or healthy mice; fresh splenocytes from 3LL or B16 mice; or CD44-depleted ALT cells derived from splenocytes of 3LL or B16 mice. Significant anti-tumor activity as shown by a reduction in primary tumor size, decreased mean number of lung metastases (Day 21), and prolonged survival was demonstrated in 3LL mice that received 3LL-derived ALT cells and in B16 mice that received B16-derived ALT cells. To test long-term immunity, adoptive transfer of subtherapeutic numbers of ex vivo activated Thy-1.2+ memory T-cells were infused into healthy congenic B6.PL Thy-1a/CY (Thy-1.1+) mice. On rechallenge with 3LL or B16 tumor, long-term tumor-specific immunity was shown to be dependent on donor Thy-1.2+ memory T-cells. These data demonstrate that tumor-specific adoptive cellular therapy and long-term tumor immunity is possible using splenocytes from tumor-bearing mice that are activated ex vivo by T3CS and is dependent on the infusion of memory T-cells. These data are consistent with clinical results in patients with metastatic melanoma and renal cell carcinoma treated with T3CS-activated lymphocytes.

Autolymphocyte therapy--I. In vivo tumour-specific adoptive cellular therapy of murine melanoma and carcinoma using ex vivo activated memory T-lymphocytes.

Autolymphocyte therapy (ALT) is tumour-specific adoptive cellular therapy of neoplastic disease based upon non-specific ex vivo activation of autologous peripheral blood lymphocytes (PBL), using the supernatant derived from a previously prepared one-way mixed lymphocyte culture (MLC). To determine the requirement for tumour antigen during the activation process, splenocytes from C57BL/6J healthy syngeneic mice (HSM) and tumour-bearing mice (TBM) were activated ex vivo using a MLC-supernatant (MLCS). Ex vivo activation was performed both in the presence (HSM splenocytes) and absence (TBM splenocytes) of a 3M KC1 syngeneic tumour-antigen (STA) extract prepared from Lewis lung (3LL) carcinoma, B16 melanoma, or normal lung. Immunophenotyping of splenocytes pre- and post-activation by MLCS plus STA or MLCS only revealed expansion of activated CD44+ (memory) T-cells. Ex vivo tumour-specific cytotoxicity was demonstrated using MLCS-activated (TBM) or MLCS + STA-activated (HSM) splenocytes against 3LL or B16 target cells. CD44+ T-cells (ALT-cells) were then infused into synegeneic 3LL and B16 TBM. Significant antitumour activity was detected in 3LL and B16 TBM receiving cells from normal mice that were activated with MLCS in the presence of 3LL or B16 STA, respectively, and in 3LL and B16 TBM receiving splenocytes from 3LL-TBM and B16-TBM, respectively, activated by MLCS alone. Infusions of 3LL-derived or B16-derived ALT-cells into HSM provided specific immunity on tumour challenge. No antitumour activity was seen in 3LL and B16 TBM receiving fresh TBM splenocytes, ALT-cells derived from HSM which were activated ex vivo using MLCS without antigen, normal lung tissue as antigen, or using MLCS-activated splenocytes without STA derived from reciprocal TBM. Ex vivo depletion of CD44+ cells or Thy-1.2+ T-cells abrogated all antitumour activity in TBM and in HSM challenged with tumour. Depletion of NK-1.1+ natural killer (NK)-cells had no effect on antitumour efficacy. These data suggest that tumour-specific adoptive cellular therapy is possible using ex vivo activated HSM splenocytes with STA, or TBM splenocytes activated ex vivo without STA, and that these antitumour effects are dependent on CD44+ memory T-cells.

Adoptive chemoimmunotherapy using ex vivo activated memory T-cells and cyclophosphamide: tumor lysis syndrome of a metastatic soft tissue sarcoma.

Adoptively transferred immune cells in combination with chemotherapeutic agents form the basis for adoptive chemoimmunotherapy (ACIT) of neoplastic disease. Autolymphocytes (ALT-cells) are ex vivo activated peripheral blood lymphocytes (PBL) from tumor-bearing hosts (TBH) that consist primarily of tumor-specific CD45RO+ (memory) T-cells. These ALT-cells combined with cimetidine (CIM) as autolymphocyte therapy (ALT), have previously been demonstrated to be a safe and active form of outpatient adoptive immunotherapy (AIT) in human TBH with metastatic renal cell cancer (RCC). We have previously described an effective ACIT protocol using ALT and cyclophosphamide (CY) for patients with relapsed and refractory non-RCC solid tumors. We now report a case of a patient with a metastatic gastric leiomyosarcoma to the liver, who developed a clinical picture consistent with a tumor-lysis syndrome (TLS), following salvage therapy for his tumor with ACIT using ALT and CY. TLS is a well-known complication resulting from the treatment of rapidly proliferating hematopoietic tumors such as Burkitt's lymphoma and acute lymphocytic leukemia. TLS has also been rarely described in chronic lymphocytic leukemia, as well as certain solid tumors such as breast cancer, small cell lung cancer, and medulloblastoma. However, there have been no previous reports of TLS occurring either secondary to immunotherapy or in sarcomas. The nature of these unusual findings is discussed.

Adoptive chemoimmunotherapy for the treatment of relapsed and refractory solid tumors using ex vivo activated memory T cells (autolymphocyte therapy) and cyclophosphamide.

Autolymphocytes (ALT cells) are ex vivo activated peripheral blood lymphocytes (PBL) from tumor-bearing hosts (TBH) that consist primarily of tumor specific CD45RO+ (memory) T cells. These ALT cells combined with cimetidine as autolymphocyte therapy (ALT) have previously been demonstrated to be a safe and active form of outpatient adoptive immunotherapy (AIT) in human TBH with metastatic renal cell cancer (RCC). To determine activity of ALT in human TBH with therapy-resistant solid tumors other than RCC and whether it was feasible to combine ALT with chemotherapy, we studied 21 patients with relapsed or primary refractory solid tumors following a study protocol of adoptive chemoimmunotherapy (ACIT) using ALT and cyclophosphamide. Twenty patients were evaluable. Five responses were seen, including two complete responses (CRs) and three partial responses (PRs). ACIT activity was noted in relapsed TBH who had responded to their previous chemotherapy and/or radiation therapy. The toxic effects of this ACIT study were minimal with no treatment-related morbidity or mortality. It appears that in some relapsed but not primary refractory solid tumors, ACIT using ALT (CD45RO+ T cells and cimetidine) together with cyclophosphamide has definite antitumor activity associated with little or no toxic effects. Further studies of ACIT in solid tumors other than RCC are justified.

Adoptive immunotherapy of hormone-refractory, stage D2 prostate cancer using ex vivo activated autologous T cells (autolymphocyte therapy): results from a pilot study.

There is no effective therapy available for stage D2 prostate cancer once patients become refractory to hormonal therapy. In a pilot study, we treated 17 patients with hormone-refractory stage D2 prostate cancer using autolymphocyte therapy, an outpatient form of adoptive immunotherapy in which patients are treated with autologous T cells that have been activated ex vivo. Feasibility and safety were documented. Transient PSA reductions up to 66% were noted, suggesting biological activity. Further studies to test the safety and efficacy of autolymphocyte therapy in the treatment of prostate cancer are warranted.

The rapid diagnosis of infectious mononucleosis using an ELISA that detects IgM antibody to a peptide component of Epstein-Barr virus nuclear antigen.

An enzyme-linked immunosorbent assay (ELISA) that detects IgM antibody to a peptide component of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) was compared with a conventional rapid heterophil antibody method for the rapid diagnosis of infectious mononucleosis. Discrepancies between the two methods were further analyzed using an indirect immunofluorescence assay to detect antibodies to EBV antigens. We evaluated 298 cases of suspected infectious mononucleosis. The ELISA was very sensitive (98.7%) and able to detect some cases (seven (9%) of 75 confirmed positives) that were negative by the rapid heterophil antibody test, but confirmed by immunofluorescence. However, approximately 17% of all positive tests could not be confirmed by EBV-specific immunofluorescence; thus, the overall positive predictive value was 83%; negative predictive value was 99.5%; and specificity was 93%. The high rate of false-positive tests makes this rapid ELISA unsuitable for the diagnosis of infectious mononucleosis.