Phase 1 study in patients with metastatic melanoma of immunization with dendritic cells presenting epitopes derived from the melanoma-associated antigens MART-1 and gp100.

Dendritic cells (DCs) have been shown to enhance anti-tumor immune responses in several preclinical models. Furthermore, DC-like function can be elicited from peripheral blood monocytes cultured in vitro with interleukin-4 and granulocyte-macrophage colony-stimulating factor. For this reason, a phase 1 study was initiated at the Surgery Branch of the National Cancer Institute to test the toxicity and biological activity of the intravenous administration of peripheral blood monocyte-derived DCs. The DCs were generated by 5- to 7-day incubation in interleukin-4 (1,000 U/mL) and granulocyte-macrophage colony-stimulating factor (1,000 U/mL) of peripheral blood monocytes obtained by leukapheresis. Before administration, the DCs were pulsed separately with the HLA-A*0201-associated melanoma epitopes MART-1(27-35) and gp-100-209-2M. The DCs were administered four times at 3-week intervals. A first cohort of patients (n = 3) was treated with 6 x 10(7) DCs and a second cohort (n = 5) with 2 x 10(8) DCs (in either case, one half of the DCs were pulsed with MART-1(27-35) and the other half was pulsed with gp-100-209-2M). In a final cohort under accrual (n = 2) 2 x 10(8) DCs were administered in combination with interleukin-2 (720,000 IU/kg every 8 hours). The recovery of DCs after in vitro culture ranged from 3% to 35% (mean, 15%) of the original peripheral blood monocytes. Administration of DCs caused no symptoms at any of the doses, and the concomitant administration of interleukin-2 did not cause toxicity other than that expected for interleukin-2 alone. Monitoring of patients' cytotoxic T lymphocyte reactivity before and after treatment revealed enhancement of cytotoxic T lymphocyte reactivity only in one of five patients tested. Of seven patients evaluated for response, one had a transient partial response with regression of pulmonary and cutaneous metastases. A relatively large number of DCs can be safely administered intravenously. The poor clinical outcome of this study perhaps could be explained by the type of protocol used for DC maturation, the route of administration, or both. For this reason, this clinical protocol was interrupted prematurely, whereas other strategies for DC preparation and route of administration are being investigated at the authors' institution.

Tumour necrosis factor receptor I (p55) is upregulated on endothelial cells by exposure to the tumour-derived cytokine endothelial monocyte- activating polypeptide II (EMAP-II).

Endothelial monocyte activating polypeptide-II (EMAP-II) is an inflammatory cytokine known to have a role in neutrophil and macrophage chemotaxis and in apoptosis. It is a tumour-derived cytokine that sensitizes tumour vasculature to the effects of systemic TNF. In order to gain insight into the mechanism by which EMAP-II sensitizes vessels to TNF, we focused on its effects on TNF receptor expression. In human umbilical vein endothelial cells (HUVEC), TNF-R1 mRNA is increased four-fold following incubation with recombinant EMAP-II. Conditioned media from cell lines known to produce high levels of EMAP-II upregulated TNF-R1 but not TNF-R2 by up to twenty-fold compared to media controls and low expressing cell lines; this effect was blocked by anti-EMAP-II antibody. Recombinant EMAP-II upregulated TNF-R1 expression by approximately six-fold. Analysis of HUVEC lysates by ELISA showed increased expression of TNF-R1 within 2 h; TNF-R2 expression was unaffected by recombinant EMAP-II. Finally, immunohistochemistry of human melanomas in vivo showed that TNF-R1 staining is increased on the vessels of tumours known to express high levels of EMAP-II compared to low EMAP-II expressing tumours. These results suggest that EMAP-II upregulates TNF-R1 expression by endothelial cells both in vitro and in vivo. This induction of TNF-R1 expression may be the mechanism by which EMAP-II sensitizes tumour endothelium to the effects of TNF leading to haemorrhagic necrosis.

Sequence-dependent enhancement of paclitaxel toxicity in non-small cell lung cancer by 17-allylamino 17-demethoxygeldanamycin.

OBJECTIVE: Overexpression of the oncogene erbB-2 contributes to chemoresistance in various malignant tumors including lung cancer. The aim of this study was to investigate whether depletion of the erbB-2 gene product (p185) by 17-allylamino 17-demethoxygeldanamycin would sensitize lung cancer cells to paclitaxel (Taxol) in vitro. METHODS: Paclitaxel cytotoxicity was evaluated in a panel of non-small cell lung cancer cell lines that expressed varying levels of p185 by means in vitro proliferation assays and 2 drug combination schedules. Cell cycle kinetics and apoptosis after exposure to paclitaxel or paclitaxel plus 17-allylamino 17-demethoxygeldanamycin were analyzed by flow cytometry. RESULTS: The 17-allylamino 17-demethoxygeldanamycin treatment efficiently depleted p185 expression in lung cancer cells. Concurrent exposure of these cells to paclitaxel and 17-allylamino 17-demethoxygeldanamycin significantly enhanced paclitaxel-mediated cytotoxicity, particularly in cells which overexpressed p185. There was a 1.3 to more than 20-fold reduction of paclitaxel 50% inhibitory concentration values in those cells that were responding positively to the drug combination. Significant induction of apoptosis was observed after treatment of cells with the combination of paclitaxel and 17-allylamino 17-demethoxygeldanamycin. The combination cytotoxic effect was only additive in cells expressing low levels of p185. In contrast, of lung cancer cells with exposure to 17-allylamino 17-demethoxygeldanamycin before combined paclitaxel and 17-allylamino 17-demethoxygeldanamycin exposure actually rendered the cells refractory to paclitaxel cytotoxicity. CONCLUSION: The compound 17-allylamino 17-demethoxygeldanamycin sensitizes non-small cell lung cancer cells expressing high levels of p185 to paclitaxel-mediated growth arrest and apoptosis. These preclinical data support the evaluation of the combination of paclitaxel and 17-allylamino 17-demethoxygeldanamycin in the treatment of patients with lung cancer whose tumors exhibit p185 overexpression.

NY-ESO-1 may be a potential target for lung cancer immunotherapy.

PURPOSE: To evaluate the frequency of NY-ESO-1 expression in cultured lung cancer cells and to determine if this cancer-testis antigen can be presented for recognition by an HLA-restricted cytolytic T-cell clone specific for NY-ESO-1. METHODS AND RESULTS: Reverse transcriptase and polymerase chain reaction amplification techniques were utilized to screen a panel of lung and esophageal cancer cell lines for expression of NY-ESO-1 encoding a recently identified cancer-testis antigen. NY-ESO-1 expression was detected in 11 of 16 small cell lung cancer lines, three of seven non-small cell lung cancer lines, and zero of 12 esophageal cancer lines. 5-Aza-2' -deoxycytidine induced expression of NY-ESO-1 in lung cancer cells. Expression of HLA-A31 by plasmid transfection or retroviral transduction enabled recognition of lung cancer cells by an HLA-A31-restricted cytotoxic T lymphocyte clone specific for NY-ESO-1. CONCLUSIONS: NY-ESO-1 expression may be analogous to MAGE gene expression in lung cancer lines in terms of frequency and mechanism of transcriptional regulation. Furthermore, NY-ESO-1 can be presented on lung cancer cells for recognition by HLA-restricted cytotoxic T lymphocytes. Further investigation is warranted to determine if NY-ESO-1 can be exploited for the immunotherapy for lung cancer.

Natural variation of the expression of HLA and endogenous antigen modulates CTL recognition in an in vitro melanoma model.

Increasing attention has been devoted to elucidating the mechanism of lost or decreased expression of MHC or melanoma-associated antigens (MAAs), which may lead to tumor escape from immune recognition. Loss of expression of HLA class I or MAA has, as an undisputed consequence, loss of recognition by HLA class I-restricted cytotoxic T cells (CTLs). However, the relevance of down-regulation remains in question in terms of frequency of occurrence. Moreover the functional significance of epitope down-regulation, defining the relationship between MHC/epitope density and CTL interactions, is a matter of controversy, particularly with regard to whether the noted variability of expression of MHC/epitope occurs within a range likely to affect target recognition by CTLs. In this study, bulk metastatic melanoma cell lines originated from 25 HLA-A*0201 patients were analyzed for expression of HLA-A2 and MAAs. HLA-A2 expression was heterogeneous and correlated with lysis by CTLs. Sensitivity to lysis was also independently affected by the amount of ligand available for binding at concentrations of 0.001 to 1 mM. Natural expression of MAA was variable, independent from the expression of HLA-A*0201, and a significant co-factor determining recognition of melanoma targets. Thus, the naturally occurring variation in the expression of MAA and/or HLA documented by our in vitro results modulates recognition of melanoma targets and may (i) partially explain CTL-target interactions in vitro and (ii) elucidate potential mechanisms for progressive escape of tumor cells from immune recognition in vivo.

Flavopiridol mediates cell cycle arrest and apoptosis in esophageal cancer cells.

Esophageal adenocarcinoma (SKGT-2, SKGT-4, and SKGT-5) and epidermoid carcinoma (HCE-4) cells containing variable retinoblastoma (Rb), cyclin D1, p16, and p53 expression patterns were exposed to the synthetic flavone, flavopiridol. The IC50 was approximately 100-150 nM for each of these cell lines. Exposure of esophageal carcinoma cells to 300 nM flavopiridol induced cell cycle arrest and apoptosis, resulting in a 90% inhibition of proliferation relative to that of nontreated cells after a 5-day exposure to the drug. Western blot analysis revealed diminution of cyclin D1, Rb, and p107 protein levels after flavopiridol exposure. Whereas cell cycle arrest and overall growth inhibition did not correlate in any obvious manner with the genotype of these cell lines, apoptosis seemed to be more pronounced in SKGT-2 and SKGT-4 cells that lack Rb expression. Pretreatment of esophageal cancer cells with 9-cis-retinoic acid did not substantially potentiate flavopiridol activity in these cell lines. Although the precise mechanism of flavopiridol-mediated cytotoxicity has not been fully defined, this drug is an attractive agent for molecular intervention in esophageal cancers and their precursor lesions; further evaluation of flavopiridol in this clinical context is warranted.

T cell-receptor V gene use by CD4+ melanoma-reactive clonal and oligoclonal T-cell lines.

Tumor-reactive CD4+ T cells can be isolated and expanded from the peripheral blood and tumor lesions of patients with melanoma. In contrast to CD8+ T cells, little is known about the antigens recognized by these CD4+ T cells. As a consequence, little is known about the diversity of the T-cell receptor (TcR) use by melanoma-reactive CD4+ T cells. To address these questions, a panel of clonal or highly oligoclonal CD4+ T-cell lines was established from a patient with metastatic melanoma. A CD4+ tumor-infiltrating lymphocyte (TIL) line was established that was highly oligoclonal and recognized only autologous melanoma cells but not allogeneic melanomas, suggesting the expression of a mutated or uniquely expressed antigen by this melanoma. The antigen recognized by the CD4+ TILs could be presented by intact melanoma cells or by autologous Epstein-Barr virus (EBV) B cells pulsed with melanoma cell lysates. A panel of CD4+ clonal and highly oligoclonal T-cell lines was isolated form peripheral blood mononuclear cells (PBMC) from this patient; these were also reactive with autologous melanoma cells or tumor extracts pulsed on autologous EBV B cells. Despite their reactivity with the autologous melanoma, we found no evidence of restricted TcR V gene use, because all six T-cell lines recognized antigen via different TcR alpha/beta rearrangements. Furthermore, there were no conserved amino acids in the CDR3 regions of these TcRs, indicating that multiple TcR clonotypes could mediate recognition of a single unique major histocompatibility (MHC) complex class II restricted melanoma antigen or that multiple MHC class II restricted melanoma antigens are expressed by the melanoma.

Identification of a T-cell receptor from a therapeutic murine T-cell clone.

Tumor-infiltrating lymphocytes (TIL) have been successfully used for the treatment of metastatic malignancies in clinical trials and in experimental animal models. Tumor-specific reactivity by TIL is mediated via receptors expressed on the surface of T cells (TcRs), which recognize tumor-associated antigens (TAA) presented in the context of MHC molecules on the surface of tumor cells. The current study was performed to identify the TcR alpha and beta chains from a tumor-specific therapeutic TIL clone that can be used to develop a preclinical animal model for genetically modifying lymphocytes and hematopoietic progenitors with TcR genes. TIL 205 was generated from a subcutaneous implant of MCA-205 fibrosarcoma and at 21 days was cloned by limiting dilution. TIL clone 8, obtained from a culture seeded at one cell/well, mediated specific lysis and specific secretion of gamma-interferon to MCA-205 and WP6, a subclone of MCA 205. No reactivity was observed against other syngeneic sarcoma lines. Anchor polymerase chain reaction analysis determined that antigen recognition by clone 8 was mediated by a TcR consisting of V alpha 3/J alpha 27 and V beta 8.2/D beta 2.1/D beta 2.4. Immunofluorescent staining with V beta subfamily specific monoclonal antibodies revealed that > 95% of the T cells in TIL clone 8 expressed V beta 8.2, confirming that TIL clone 8 was indeed a clone. In contrast, approximately 30% of the T cells in the parental TIL 205 expressed V beta 8.2. The transfer of as few as 500,000 TIL clone 8 cells in conjunction with the systemic administration of recombinant human interleukin-2 mediated regression of established 3-day WP6 lung metastases. Thus, clone 8 recognizes a biologically relevant tumor rejection antigen, making the V alpha 3/J alpha 27-V beta 8.2/D beta 2.1/J beta 2.4 TcR isolated from this clone useful as a probe for cloning the tumor-rejection antigen in the WP6 tumor as well as modeling, in mice, the TcR-based gene therapies being developed for humans.

Tumor-infiltrating lymphocytes from human ovarian cancer patients recognize autologous tumor in an MHC class II-restricted fashion.

PURPOSE: To search for tumor-specific in vitro reactivity by lymphocytes derived from patients with ovarian carcinoma. METHODS: Tumor-infiltrating lymphocytes (TIL) were derived from primary or metastatic solid tumors and tumor-associated lymphocytes (TAL) were derived from ascites from 13 patients with ovarian cancer. TIL or TAL were cultured for approximately 30 to 60 days and studied for phenotype, cytotoxicity, and cytokine secretion in response to autologous tumor stimulation. RESULTS: Twenty-nine bulk TIL or TAL cultures were successfully established from 10 patients using various culture conditions. Thirteen cultures were predominantly CD4+ and 16 were mainly CD8+. In contrast to reports by others, none of the cultures tested were specifically lyric for autologous tumor. Five predominantly CD4+ bulk TIL (from four patients) preferentially secreted tumor necrosis factor-alpha and granulocyte macrophage-colony stimulating factor when stimulated with autologous tumor and not when stimulated by autologous Epstein Barr virus-B cells, fibroblasts, peripheral blood mononuclear cells, or allogeneic HLA matched or mismatched stimulators. This cytokine secretion was found to be MHC class-II restricted in three patients because it was inhibited by the anti-MHC class-II antibody IVA12 and the HLA-DR specific antibody L243. CONCLUSION: We believe these data are the first to suggest that tumor reactive CD4+ lymphocytes exist in some ovarian cancer patients. This finding may be useful in the development of novel immunotherapies for these patients.

Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes.

MHC class I antigen expression is necessary for CD8+ T-cell-mediated recognition of tumors. Recently, several mechanisms leading to loss or decreased expression of MHC antigens on the tumor cell surface have been described that may account for tumor escape from immune recognition. It is yet unknown whether tumor recognition by CTL occurs at a threshold amount of MHC molecules or correlates with the level of HLA-allele expression. In this study, a model was developed in which clones derived from the 624-MEL melanoma cell line and expressing varying amounts of HLA-A2 molecules were lysed in a standard 51Cr release assay by an HLA-A2-restricted CTL clone (A42) or a bulk culture of tumor-infiltrating lymphocytes. The A42 clone and the tumor-infiltrating lymphocyte culture were characterized previously as specifically recognizing the melanoma antigen MART-1(27-35) peptide. A marked heterogeneity in the susceptibility to lysis by A42 was observed in tumor clones and was not due to heterogeneous expression of MART-1 by the clones or loss of accessory molecules involved in the lymphocyte-target interaction. Lysis by A42 and by the tumor-infiltrating lymphocyte culture significantly correlated with the level of HLA-A2 expression, evaluated as mean channel number of fluorescence by flow cytometry (P < 0.001). Transfection of an HLA-A2-negative clone (624.28) with the HLA-A2.1 gene produced a panel of clones expressing different levels of HLA-A2, the lysis of which was highly correlated with the expression of HLA-A2 (P < 0.001). The addition of exogenous MART-1(27-35) peptide enhanced lysis of clones expressing intermediate amounts of HLA-A2 but did not affect clones with high expression. These data suggest that the number of HLA molecules present on the surface of tumor cells can quantitatively affect their lysis by CTL in situations with borderline amounts of peptide and/or MHC.

Helper T cells infiltrating human renal cell carcinomas have the phenotype of activated memory-like T lymphocytes.

Human renal cell carcinomas are characterized by an inflammatory infiltrate containing many T lymphocytes. Attempts to grow T cells from such tumors by culture in interleukin (IL)-2 have yielded heterogeneous populations of cells with functional characteristics typical of lymphokine-activated killer cells obtained by similar culture of cells from peripheral blood mononuclear cells. We examined a panel of surface markers expressed on T lymphocytes to determine if the CD4+ T cells infiltrating human renal cell carcinomas are different from those in peripheral blood mononuclear cells. By flow cytometry analysis the CD4+ T cells in a panel of freshly digested human renal cell carcinoma primary and metastatic tumors expressed the activation markers CD69 and HLA-DR and manifested an increase in CD45RO and a reciprocal decrease in CD45RA expression as compared with peripheral blood CD4+ T cells. This suggests that CD4+ T cells infiltrating renal cell carcinomas are activated and have encountered antigen. However, the expression of the IL-2R alpha chain (CD25) was not different in tumor-infiltrating CD4+ T cells and peripheral blood CD4+ T cells, suggesting that T cells infiltrating human renal cell carcinomas may have a block in proliferative capacity. The general failure of cultured tumor-infiltrating lymphocyte (TIL) from renal cell carcinoma to demonstrate tumor-specific reactivity may be due to the failure of such cells to grow in IL-2.

Loss of HLA haplotype and B locus down-regulation in melanoma cell lines.

The expression of HLA class I molecules on tumor cells is vital for CD8+T cell recognition of tumor Ags. Loss of HLA class I Ag expression as a result of defective beta 2-microglobulin genes has been described in melanoma cells. To further evaluate mechanisms of tumor escape, HLA class I Ag expression was compared in 24 metastatic melanoma cell lines and 20 melanocyte strains by FACS analysis with use of allele-specific mAbs. Total loss of HLA class I Ag expression was not noted; instead, two relatively common phenomena were identified: 1) A variable degree of expression of HLA-B Ags by melanoma cell lines and melanocytes; however, HLA-A Ags were consistently expressed in all cell types. Furthermore, HLA-B locus Ag expression was detected in vivo in only one of six frozen section specimens obtained from six patients having metastatic melanoma. 2) Loss of allelic expression was noted in two of 14 HLA-A2 (14%) and one of three HLA-A29 (33%) melanoma cell lines and included a full haplotype, which suggests loss of a genomic fragment. Allele-specific PCR amplification demonstrated deletion of genes in linkage disequilibrium within the MHC class II, III, and I regions. Aberrations of HLA class I expression in tumor lines should be considered when assessing MHC-restricted phenomena in in vitro models.

Bone marrow transplantation in miniature swine: IV. Development of myeloablative regimens that allow engraftment across major histocompatibility barriers.

Studies of the myeloablative regimens capable of permitting successful BMT across MHC barriers in miniature swine have been performed. To minimize graft-versus-host disease (GVHD), engraftment was studied in the F1-->P combination (i.e., MHC homozygous ["parental"] swine receiving bone marrow from one-haplotype matched MHC heterozygous ["F1"] donors). Animals given total body irradiation (TBI) up to 1100 cGy, 10 cGy/min, in a single dose failed to engraft. Increasing the dose rate led to unacceptable extramedullary toxicity without improving engraftment. Eleven different fractionated TBI regimens were tested in this F1-->parent model. At all of the dose rates tested, a total dose of less than 1000 cGy was insufficient for engraftment, and a total dose of 1400 cGy led to unacceptable toxicity. Between these extremes, a window was defined in which engraftment could be obtained without unacceptable extramedullary toxicity utilizing 2 equally divided fractions of TBI delivered 24 hr apart. The addition of 50 mg/kg cyclophosphamide i.v. to fractionated TBI (1150 cGy total dose [500 + 650]) also permitted engraftment, with decreased incidence of interstitial pneumonitis as compared to fractionated TBI (1300 cGy total dose [650 x 2]). Both of these regimens were also confirmed to permit engraftment between heterozygous donors and recipients sharing a single common haplotype ("F1-->F1"). The regimen of 1300 cGy (650 x 2) also permitted engraftment in completely MHC mismatched BMT, but with subsequent death from GVHD. These studies of the myeloablative regimens permitting engraftment across defined MHC barriers in miniature swine provide a basis for further studies of allogenic BMT and GVHD in this large animal preclinical model.

Phase I trial of subcutaneous interleukin-6 in patients with advanced malignancies.

PURPOSE: Based on preclinical evidence in murine models that interleukin-6 (IL-6) mediates regression of metastatic tumors, we performed a phase I study of recombinant human IL-6 in patients with refractory advanced malignancies to determine its pharmacokinetics, toxicities, and possible immunologic and antitumor effects. PATIENTS AND METHODS: Recombinant IL-6 was administered as a single subcutaneous dose daily for 7 days, with 7 days off therapy followed by another 7 days of IL-6. Doses were escalated in cohorts of three patients starting at 3 micrograms/kg/d, provided that toxicity at the preceding dose level was not dose-limiting. Dose-limiting toxicity was defined as grade III or IV major organ toxicity that did not resolve to grade II or less in 24 hours after stopping IL-6, using the National Cancer Institute Common Toxicity Criteria. Patients were treated with 3, 10, and 30 micrograms/kg/d IL-6 subcutaneously. RESULTS: Three patients each were treated at the 3- and 10-micrograms dose levels. Two of five patients treated with 30 micrograms/kg/d IL-6 subcutaneously had grade III major organ toxicity that required IL-6 therapy to be discontinued. All patients experienced fever, chills, and minor fatigue. Significant increases in C-reactive protein (CRP), fibrinogen, platelet counts, and lymphocyte IL-2 receptor levels were seen in patients at the 10- and 30-micrograms/kg dose levels. Decreases in albumin and hemoglobin were observed, particularly at the 30-micrograms/kg dose level. The half-life (T1/2 beta) was 4.2 hours, with a peak IL-6 level at 5 hours. No antitumor responses were seen. CONCLUSION: A safely tolerated dose of daily subcutaneous IL-6 is 10 micrograms/kg, with hepatotoxicity and cardiac arrhythmia being the dose-limiting toxicities at 30 micrograms/kg. Phase II trials of IL-6 administered subcutaneously daily for at least 7 days for two cycles with an intervening week of rest are recommended for phase II trials. However, patients with extensive replacement of liver by tumor and abnormal liver functions should receive IL-6 therapy with caution.

In vitro and in vivo effects of recombinant human interleukin-2 in naive miniature swine.

Recent data in mice have shown that early administration of recombinant human interleukin-2 (rIL-2) provides significant protection from lethal graft-versus-host disease. Because of the potential clinical importance of these findings, it will be important to assess the effectiveness of this therapy in a large animal preclinical bone marrow transplantation model. We report here our initial studies of the in vitro and in vivo effects of rIL-2 in miniature swine. In vitro 4-day cultures of pig peripheral blood lymphocytes (PBL) in complete medium containing rIL-2 at 1,000 U/ml resulted in optimal proliferation and generation of lymphokine-activated killer (LAK) cells. A pig-mouse hybridoma cell line was found to be highly sensitive as a LAK cell target. Two naive pigs received 20,000 U/kg and 2 pigs received 100,000 U/kg of rIL-2 intravenously twice a day for 4 days. No clinical symptoms were seen during or after administration at the lower dose while both high dose-treated animals showed generalized erythema from days 2 to 4, and one showed mild diarrhea during this period. The disappearance of IL-2 activity from the serum showed two components: (1) an initial fast component with a half-time of approximately 10 min and (2) a slow component with a half-time of approximately 60 min. LAK cell precursors disappeared from the peripheral circulation by 6 min after rIL-2 administration and began to recover by 6 h in the low dose recipients and only after 12 h in the high dose recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

Successful induction of long-term specific tolerance to fully allogeneic renal allografts in miniature swine.

Major histocompatibility complex class II matching is of overwhelming importance for achieving tolerance to kidney transplants (KTX) in miniature swine. When class II antigens are matched, long-term specific tolerance across complete MHC class I antigen barriers can uniformly be induced by a 12-day perioperative course of cyclosporine. This same regimen is ineffective in fully MHC-mismatched combinations. We hypothesized that initial induction of tolerance to kidney donor class II antigens by bone marrow transplantation might allow tolerance to be induced to a subsequent fully allogeneic KTX in combination with CsA therapy. We report here the results of such fully allogeneic KTX performed in 4 recipients of prior single-haplotype class II-mismatched BMT. All animals received KTX from donors class II matched to the BMT donor and received a 12-day course of intravenous CsA (10 mg/kg/day). All four animals have maintained normal serum creatinine values (less than 2.0 mg/dl) for greater than 200 days posttransplant. Specific hyporesponsiveness to both BMT and KTX donor-type MHC antigens was found in mixed lymphocyte culture and cell-mediated lympholysis assays. Compared with third-party grafts, significantly prolonged survival of BMT donor-specific (P = 0.031) and KTX donor-specific (P = 0.031) skin grafts was observed. These results demonstrate that induction of tolerance to class II antigens by BMT allows a short course of CsA to induce specific tolerance to fully allogeneic renal allografts.

Induction of kidney transplantation tolerance across major histocompatibility complex barriers by bone marrow transplantation in miniature swine.

Previous studies from our laboratory have shown that permanent lymphohematopoietic chimerism can be induced in MHC-disparate miniature swine by bone marrow transplantation after lethal total-body irradiation. The purpose of the present study was to determine in this large animal model whether such chimerism would lead to permanent tolerance to a vascularized allograft without a requirement for exogenous immunosuppression. Eight miniature swine that had received MHC-mismatched BMT more than five months earlier underwent kidney transplantation (KTx) from a donor MHC matched (n = 5) or MHC mismatched (n = 3) with the BMT donor. All animals had regained in vitro responsiveness to third-party MHC antigens, as measured by mixed lymphocyte reaction (MLR), before KTx but remained nonresponsive to MHC antigens of the BMT donor and self. All three animals that received KTx mismatched for BMT donor MHC rejected promptly (mean survival time 7.0 days). Of the five animals that received KTx matched for BMT donor MHC, four showed no evidence of rejection and have functioning KTx greater than 200 days after KTx. The fifth animal had excellent renal function for 60 days but then developed a slowly rising BUN and serum creatinine, and died 75 days after KTx. The course of this animal's rejection is consistent with that previously described for rejection due to minor antigen disparities. The difference in survival of KTx matched or mismatched for the MHC of the BMT donor was statistically significant (P = 0.0062). The survival of KTx matched for the MHC of the BMT donor was significantly different from that of control animals without BMT receiving KTx mismatched for MHC (P = 0.0018). We therefore conclude that BMT is an effective means for induction of tolerance to an MHC mismatched KTx in this large animal model.

In vivo treatment with antiporcine T cell antibodies.

The effects of in vivo administration of monoclonal antibodies directed against the two major mature T cell subsets of miniature swine have been examined. Antibody 76-2-11, specific for the porcine CD8 cell-surface antigen, caused elimination of CD8+ T cells from the peripheral lymphocyte pool by 4-6 days following a single intravenous infusion. This depletion persisted while excess antibody was detectable in the serum and CD8+ cells returned rapidly to normal levels following the disappearance of serum antibody. In vitro functional assays performed on PBL during the period of depletion showed no effect on proliferative responses but a profound inhibition of ability to generate CTL. In contrast, administration of monoclonal antibody 74-12-4, specific for CD4+ porcine T lymphocytes, caused no consistent change in the level of CD4+ cells in the peripheral lymphocyte pool. By flow microfluorometry, CD4+ peripheral lymphocytes were found to be coated with the antibody, and there also was excess antibody in the serum for at least 4 days in two of the three animals treated. No effects on in vitro proliferative responses or generation of CTL in vitro were observed, even during the period of excess circulating antibody. Treatment with both antibodies was tolerated well by all animals. These findings are similar to those that have been found in clinical studies of monoclonal antibody treatment and suggest that the pig will provide a large animal preclinical model in which the effects of in vivo treatment with monoclonal antibodies on parameters of transplantation immunity can be tested.