Search

Targeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form of CD43 expressed by hematologic malignancies, is a recently identified target and antibodies interacting with CD43s may have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was derived from a high-risk AML patient who successfully cleared leukemia after allogeneic stem cell transplantation. Here we observed that AT1413 binds also to certain non-hematopoietic tumor cells, particularly melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody-dependent cellular cytotoxicity against short-term cultured patient-derived melanoma samples. However, AT1413 was unable to affect the growth of melanoma cells in vivo. To increase the efficacy of AT1413 as a therapeutic antibody, we generated two different formats of bispecific T-cell engaging antibodies (TCEs): one binding bivalently (bTCE) and the other monovalently (knob-in-hole; KiH) to both CD43s and CD3ε. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with differences in potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth in these mice. These data indicate a broad therapeutic potential of AT1413 that includes AML and CD43s-expressing solid tumors that originate from CD43-negative tissues.

To investigate prognostic factors in patients with recurrent cervical cancer after treatment of early-stage disease in order to identify high-risk patients who might benefit from alternative treatment strategies.The authors retrospectively analyzed clinical and pathology data from 130 recurrent cervical cancer patients after surgical treatment for early-stage disease. Patients were compared with a recurrence-free control group matched for age, FIGO Stage, and adjuvant treatment. Univariate and multivariate Cox regression analyses were performed to determine prognostic factors for recurrence and survival.Of 889 patients, 130 (14.6%) developed recurrent disease after primary treatment for early-stage cervical cancer. Local or loco-regional metastasis was observed in 45%, distant metastasis in 31%, and combined pelvic and distant metastasis in 24%. Median survival after recurrence was 12 months (range 1-107 months). Median five-year survival was 96% in the control group and 29% in the recurrence group. Tumor size ≥ 40 mm and lymph node metastasis were independent unfavorable prognostic factors for overall survival (OS) and disease-free survival (DFS). The number of positive lymph nodes (≥ one) and bilateral occurrence of pelvic lymph node metastasis were associated with adverse clinical outcome.Tumor size ≥ 40 mm and lymph node metastasis were independent unfavorable prognostic factors in surgically treated, early-stage cervical cancer patients. The combination of these factors was particularly associated with recurrence. Future trials should focus on the role of alternative adjuvant treatment strategies in patients at high risk of recurrent disease (e.g., by chemotherapy, immunotherapy or combinations thereof).

Bispecific Abs (BsAb) represent a novel format of immunotherapy, recognizing immune effector cells (e.g., T cells), on the one hand, and target cells (e.g., tumor cells), on the other hand. To be successful, cross-linking of the two cell types is necessary for effector cell activation and subsequent killing of the malignant target cells. We asked the question, whether CTL that were incubated with the BsAb aCD3 x aCD19 and malignant B cells and activated to kill the malignant B cells were still able to eliminate their natural target cells (e.g., virus-infected autologous body cells). To test this, HLA-A*0201-restricted, influenza-specific CTL were incubated with BsAb- and HLA-A*0201-positive B lymphoid tumor cells in combination with HLA-A*0201-positive, virus-infected non-B lymphoid cells as natural target cells. The results showed that even in the presence of BsAb and high amounts of tumor B cells, CTL were still capable of eliminating the virus-infected non-B lymphoid target cells; actually, CTL recognized and eliminated the homologous original target cells preferentially.

Immunotherapy of usual vulvar intraepithelial neoplasia (uVIN) is promising; however, many patients still fail to show clinical responses, which could be explained by an immune escape through alterations in human leukocyte antigen (HLA) expression. Therefore, we analyzed a cohort of patients with a primary (n = 43) and subsequent recurrent uVIN lesion (n = 20), vaccine-treated uVIN patients (n = 12), patients with human papillomavirus (HPV)-induced vulvar carcinoma (n = 21) and healthy controls (n = 26) for the expression of classical HLA-class I/II and nonclassical HLA-E/-G and MHC class I chain-related molecule A (MICA). HLA-class I was downregulated in 70% of uVIN patients, including patients with a clinical response to immunotherapy. Downregulation of HLA-class I is probably reversible, as only 15% of the uVIN cases displayed loss of heterozygosity (LOH) and HLA-class I could be upregulated in uVIN keratinocyte cultures by interferon γ. HLA-class I downregulation is more frequently associated with LOH in vulvar carcinomas (25-55.5%). HLA-class II was found to be focally expressed in 65% of uVIN patients. Of the nonclassical molecules, MICA was downregulated in 80% of uVIN whereas HLA-E and -G were expressed in a minority of cases. Their expression was more prominent in vulvar carcinoma. No differences were found between the alterations observed in paired primary and recurrent uVIN. Importantly, downregulation of HLA-B/C in primary uVIN lesions was associated with the development of recurrences and progression to cancer. We conclude that downregulation of HLA is frequently observed in premalignant HPV-induced lesions, including clinical responders to immunotherapy, and is associated with worse clinical outcome. However, in the majority of cases downregulation may still be reversible.

One of the hallmarks of cancer is the influx of myeloid cells. In our study, we investigated the constitution of tumor-infiltrating myeloid cells and their relationship to other tumor-infiltrating immune cells, tumor characteristics and the disease-specific survival of patients with cervical cancer (CxCa). Triple-color immunofluorescence confocal microscopy was used to locate, identify and quantify macrophages (CD14), their maturation status (CD33) and their polarization (CD163) in a cohort of 86 patients with cervical carcinoma. Quantification of the numbers of myeloid cells revealed that a strong intraepithelial infiltration of CD14+ cells, and more specifically the population of CD14+CD33-CD163- matured M1 macrophages, is associated with a large influx of intraepithelial T lymphocytes (p = 0.008), improved disease-specific survival (p = 0.007) and forms an independent prognostic factor for survival (p = 0.033). The intraepithelial CD8+ T-cell and regulatory T-cell (Treg) ratio also forms an independent prognostic factor (p = 0.010) and combination of these two factors reveals a further increased benefit in survival for patients whose tumor displays a dense infiltration with intraepithelial matured M1 macrophages and a high CD8 T-cell/Treg ratio, indicating that both populations of immune cells simultaneously improve survival. Subsequently, we made a heatmap including all known immune parameters for these patients, whereby we were able to identify different immune signatures in CxCa. These results indicate that reinforcement and activation of the intratumoral M1 macrophages may form an attractive immunotherapeutic option in CxCa.

The impact of the MHC class I peptide binding stability on the immunogenicity of particular peptide Ags in class I-restricted cytotoxic T lymphocyte responses is not clearly established. Therefore, we have determined the dissociation rate of each peptide from MHC class I at 37 degrees C and compared this to that of a consensus CTL epitope. Newly defined immunogenic peptides formed relatively stable MHC-peptide complexes as shown by their low dissociation rates, whereas nonimmunogenic peptides displayed high dissociation rates. In addition virtually all previously described HLA-A*0201-restricted T cell epitopes showed low dissociation rates. Furthermore, we show that the immunogenicity of HIV-1-derived peptides can be predicted more accurately by their dissociation rate than by the MHC class I binding affinity. Selection of peptides based on affinity and their dissociation rate leads to a more precise identification of candidate CTL epitopes than selection based on affinity alone. These results help to understand why some peptides are recognized by CTL and, along with detailed knowledge of protein processing rules, therefore have important implications for the selection of peptides in peptide-based vaccines.

SUMMARY Despite the well described infiltration of cells of the cellular immune system in vasculitic lesions and the granuloma formation in patients with WG, the role of T cell-mediated autoimmunity in WG is not clear. Reports of T cell proliferation in response to neutrophil azurophilic granule proteins are contradictory. In this study we have assessed the proliferation of T cells of WG patients to purified proteinase 3 (PR3) and to total azurophilic granule proteins in two different assays. In addition to the classical proliferation assay with isolated peripheral blood mononuclear cells, we have used a whole blood proliferation assay. In both assays we found proliferative responses to PR3 in patients with WG. The number of patients reacting to the azurophilic granule extract was higher than the patients reacting to the purified PR3, suggesting that other autoantigens may also be involved. We have identified epitopes of PR3 that may be potential targets of class I-restricted T cell responses in the context of HLA-A*0201, the most common MHC class I molecule. These epitopes were determined by the binding of synthetic PR3 peptides to HLA-A*0201 on the antigen-processing defective cell line, T2. In addition, T cell lines were established from tissue biopsies, obtained from WG patients, and assessed for cytolytic reactivity against T2 cells, preloaded with synthetic PR3 peptides. We conclude that T lymphocytes of WG patients have increased proliferative responses to purified PR3 and to a larger extent to non-fractionated proteins of azurophilic granules of polymorphonuclear neutrophilic leucocytes (PMN).

The design of the human papillomavirus (HPV) infection cycle is tightly fitted to the differentiation program of its natural host, the keratinocyte. This has important consequences for the role of antigen-presenting cells in the priming of antiviral immunity. The confinement of HPV infection to epithelia puts the epithelial dendritic cell, the Langerhans cell (LC), in charge of the induction of T cell-dependent immunity. Because HPV-infected keratinocytes cannot reach the regional lymphoid organs, and HPV-infection of LCs does not result in viral gene expression, priming of antiviral T cells exclusively depends on cross-presentation of viral antigens by the LC. Sensitization of the immune system in the regional lymphoid organs elicits systemic anti-HPV immunity as well as intraepithelial immune surveillance by memory-type intraepithelial T cells and locally produced antibodies. The high rate of spontaneous rejections of high-risk HPV-infections and HPV-positive premalignant lesions indicates that in general the LC-driven antigen presentation machinery is capable of raising an effective immune defense against HPV. Epidemiological studies also reveal that a decrease in the vigilance of the immune system is readily exploited by HPV to escape immune destruction, resulting in persistent infections and development of HPV-positive cancers. In view of the inherent antigenicity of HPV, immune intervention strategies constitute a promising approach for both the prevention and the therapeutic treatment of HPV-induced diseases. Importantly, the mechanisms that govern the induction and effector phases of the intraepithelial immune surveillance against HPV must be taken into account when designing such strategies.

Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine-induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA-typed healthy donors, we identified the central part of HPV16 E7 (E7(41-72)) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E7(50-62), DR3/E7(43-77), DQ2/E7(35-50)). In a parallel approach, employing IFN-gamma ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16+ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16+ subjects did not display any E7-specific type 1 cytokine-producing T-cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16-specific T-cell immunity using the ELISPOT assay described, as well as for HPV-specific vaccination of individuals with HPV+ lesions.

The human epithelial mucin MUC1 is over-expressed in more than 90% of carcinomas of the breast, ovary, and pancreas as well as in some other tumours, making it a potential target for tumour immunotherapy. We have identified several MUC1-derived peptides mapping outside the variable number tandem repeat region that comply with the peptide-binding motif for HLA-A*0201 and that become processed into stable major histocompatibility complex-peptide complexes as assessed by in vitro assays. In A2/K(b) transgenic mice, 3 peptides, namely MUC(79-87) (TLAPATEPA), MUC(167-175) (ALGSTAPPV) and MUC(264-272) (FLSFHISNL) elicit peptide-specific cytotoxic T lymphocyte (CTL) immunity, which protects these mice against a challenge with MUC1, A2/K(b)-expressing tumour cells. These peptides therefore represent naturally processed MUC1-derived CTL epitopes that could be used as components in peptide-based vaccines and for the analysis of anti-MUC1 CTL responses in A*0201-positive patients with MUC1-expressing tumours.

Colorectal carcinoma is commonly associated with mutation and overexpression of p53, making this antigen a potential target for immune intervention. We analyzed humoral and proliferative immunity against p53 in the blood of patients with resected primary colorectal cancer. The majority of these patients displayed anti-p53 T helper (Th) immunity in the absence of measurable p53 specific antibody levels. The Th responses were long-lasting since they could be detected up to several years after resection of the primary tumor. In a number of cases the Th responses were highly sensitive, reflected by the recognition of naturally processed p53 protein. Our data argue that boosting of these responses in patients with minimal residual disease through p53-specific vaccination, may be employed for improving the chance of disease-free survival of these patients.

CD4+ Th cells play an important role in the induction and maintenance of specific T cell immunity. Indications for a protective role of CD4+ T cells against HIV-1 infection were found in subjects who were able to control HIV-1 viremia as well as in highly HIV-1-exposed, yet seronegative, individuals. This study describes the identification of an HIV-1-specific Th epitope that exhibits high affinity binding as well as high immunogenicity in the context of at least four different HLA-DR molecules that together cover 50-60% of the Caucasian, Oriental, and Negroid populations. This HIV-1 reverse transcriptase-derived peptide (RT171-190) is highly conserved among different HIV-1 isolates. Importantly, stimulation of PBL cultures from HIV-1 seronegative donors with this peptide resulted in Thl-type lymphocytes capable of efficient recognition of HIV-1-pulsed APCs. Taken together, these data indicate that peptide RT171-190 constitutes an attractive component of vaccines aiming at induction or enhancement of HIV-1-specific T cell immunity.

Bispecific Abs (BsAb) represent a novel format of immunotherapy, recognizing immune effector cells (e.g., T cells), on the one hand, and target cells (e.g., tumor cells), on the other hand. To be successful, cross-linking of the two cell types is necessary for effector cell activation and subsequent killing of the malignant target cells. We asked the question, whether CTL that were incubated with the BsAb aCD3 x aCD19 and malignant B cells and activated to kill the malignant B cells were still able to eliminate their natural target cells (e.g., virus-infected autologous body cells). To test this, HLA-A*0201-restricted, influenza-specific CTL were incubated with BsAb- and HLA-A*0201-positive B lymphoid tumor cells in combination with HLA-A*0201-positive, virus-infected non-B lymphoid cells as natural target cells. The results showed that even in the presence of BsAb and high amounts of tumor B cells, CTL were still capable of eliminating the virus-infected non-B lymphoid target cells; actually, CTL recognized and eliminated the homologous original target cells preferentially.

HLA-B57 has been shown to be strongly associated with slow disease progression in human immunodeficiency virus type 1 (HIV-1)-infected patients from the Amsterdam Cohort. Since HIV-1-specific CTL can control and eliminate virus-infected cells, we sought to characterize the dominant HLA-B57-restricted CTL responses at the epitope level. It was found that HLA-B57-restricted CTL responses were targeted at multiple proteins of HIV-1, with CTL specific for Gag and RT being the most pronounced. Gag-specific CTL recognized peptides ISPRTLNAW (aa 147-155) and STLQEQIGW (aa 241-249), which had previously been reported as HLA-B57-restricted. The RT-specific CTL response in one long-term survivor studied in great detail persisted for > 10 years and was dominated by HLA-B57-restricted CTL that recognized the newly defined epitope IVLPEKDSW (RT(LAI), aa 244-252). This epitope could be recognized in the context of both HLA-B*5701 and HLA-B*5801. Interestingly, three epitope variants of IVLPEKDSW were observed, which coincided with the strongest detectable CTL response to RT. One variant (T2E7) was not recognized by IVLPEKDSW-specific CTL despite the fact that this variant bound to HLA-B*5701 with a similar affinity as the index peptide. Finally, only viruses which contained the epitope index sequence were obtained suggesting efficient virus control by CTL. In conclusion, we report the characterization of dominant HIV-1 Gag- and RT-derived, HLA-B57-restricted CTL epitopes which are associated with longer time to AIDS. Further characterization of CTL responses restricted by HLA-B57 and other protective HLA alleles may contribute to the development of effective AIDS vaccines.

MHC-class-I-restricted cytotoxic T lymphocytes (CTL) specific for tumor-associated antigens expressed by malignant cells are important components of the immune response against cancer. Recently, tumor-specific CTL could be generated in vitro, with responding lymphocytes from the blood of healthy blood donors. In the present study, we confirm that peptide-specific stimulation in vitro can induce high-affinity CTL capable of recognizing tumor cells expressing the appropriate tumor antigen. These tyrosinase-specific CTL display a restricted usage of TCRAV and TCRBV gene segments but of diverse CDR3 regions, resulting in a distinct fine-specificity for each CTL clone. This suggests that, similar to in vivo priming, peptide-pulsed APC are capable of stimulating a T-cell response in vitro expressing a limited TCR repertoire against autologous tumors. The generated CTL can recognize their target structure with high affinity, and this correlates in part with tumor-cell lysis. This methodology may be used to treat melanoma patients with infusion of ex vivo-induced and -expanded CTL.

MAGE-2 is expressed in many tumors, including melanoma, laryngeal tumors, lung tumors and sarcomas, but not in healthy tissue, with the exception of testis. Thus, MAGE-2-derived peptides that bind to HLA class I molecules and elicit cytotoxic T lymphocyte (CTL) responses could be of significant therapeutic importance. In this study, we show that several MAGE-2-derived peptides bind with high affinity to HLA-A*0201. Three of them form complexes with HLA-A*0201 that are stable at 37 degrees C and are immunogenic in HLA-A*0201Kb transgenic mice. Moreover, CTLs against 2 of them (M2 112-120, and M2 157-166) specifically recognize cells that express both the MAGE-2 protein and HLA-A*0201Kb. These 2 peptides are processed and presented in the context of HLA-A*0201. Therefore, these peptides are candidate components in peptide-based vaccines for the treatment and prevention of several types of MAGE-2-expressing cancers.

A small group of HIV-1-infected subjects who either do not progress to AIDS or progress only slowly have sustained HIV-1-specific CTL responses. It has been suggested that the specificities of these responses differ from the CTL responses of rapid progressors due to recognition of epitopes that are under structural or functional constraints. We have, in this respect, studied the CTL response to reverse transcriptase (RT) in long term survivors (LTS) and in HIV-1-infected individuals who progressed to AIDS within 3 to 6 yr. Both LTS and progressors displayed vigorous RT-specific CTL responses of comparable magnitude during the asymptomatic phase. From each individual at least two CTL lines were obtained from blood samples drawn at different time points during follow-up. A total of 19 CTL lines recognized nine different RT-derived epitopes. CTL obtained from progressors recognized epitopes with a similar degree of amino acid conservation as epitopes targeted by CTL from LTS. Furthermore, five of seven epitopes were recognized by both LTS and progressors. Moreover, one of the epitopes recognized by progressors contained the highly conserved YMDD motif that is essential for RT activity. In conclusion, our data imply that neither the magnitude nor the specificity of HIV-1-specific CTL against RT is a major cause of a more protracted course of disease.

The MHC class-I binding affinity of an epitope is an important parameter determining the immunogenicity of the peptide-MHC complex. In order to improve the immunogenicity of an epitope derived from melanocyte lineage-specific antigen gp100, we performed amino-acid substitutions within the epitope and assayed both HLA-A*0201 binding and CTL recognition. Anchor replacements towards the HLA-A*0201 peptide-binding motif gave rise to peptides with higher HLA-A*0201 binding capacity compared to the wild-type epitope. In addition, several of the gp100 154-162 epitope-analogues were more efficient at target-cell sensitization for lysis by anti-gp100 154-162 CTL compared to the wild-type epitope. These altered gp100 154-162 epitopes were subsequently tested for their capacity to induce CTL responses in vivo using HLA-A*0201/Kb transgenic mice, and in vitro using HLA-A*0201 + donor-derived lymphocytes. Interestingly, the peptide-specific CTL obtained, which were raised against the different gp100 154-162 epitope-analogues, displayed cross-reactivity with target cells endogenously processing and presenting the native epitope. These data demonstrate that altered epitopes can be exploited to elicit native epitope-reactive CTL. The use of epitope-analogues with improved immunogenicity may contribute to the development of CTL-epitope based vaccines in viral disease and cancer.

A peptide-binding assay employing the HLA class I molecules on intact human B cells is described. The peptide antigens are stripped from the HLA class I molecules by mild acid treatment, after which the cells are incubated with a FL-labeled reference peptide together with different concentrations of the peptide of interest. The effectiveness by which the latter peptide competes for binding to the HLA class I molecules is assayed by measuring the amount of HLA-bound FL-labeled reference peptide with FACscan analysis. The assay is easy to perform because there is no need to purify HLA class I molecules, or to transfect cells with HLA class I molecules, and no radioactive label is used. Moreover, large panels of HLA-typed human B-cell lines are available as tools for peptide binding to a vast array of HLA molecules. The binding assay was optimized and validated with peptides of known binding capacity to either HLA-A*0201 or HLA-A*0301. The kinetics of peptide binding in this assay were shown to be comparable to that in assays employing soluble HLA class I molecules. Application of the assay in the search for potential HLA-A*0301 restricted CTL epitopes, derived from HIV-1 polymerase, resulted in the identification of five high-affinity binding peptides.

To identify novel major histocompatibility complex (MHC) class I-restricted cytotoxic T-lymphocyte (CTL) epitopes conserved in HIV-1.Potential conserved CTL epitopes were selected using a predictive computer algorithm based on a human leukocyte antigen (HLA)-A*0201 peptide-binding motif and tested for actual binding to the human processing defective cell line 174.CEM T2 (T2). Hence, the amino-acid sequences of 14 full-length sequenced HIV-1 strains were analysed. An in vitro primary peptide-specific human CTL response was induced with responding lymphocytes of an HIV-1-seronegative donor. Responding T cells were cloned by limiting dilution and tested for their ability to recognize naturally processed antigen in a 51Cr-release assay using recombinant vaccinia-HIV protein-infected B-lymphoblastoid cells (B-LCL) as target cells.The analysis of peptides bearing the HLA-A*0201 motif for conservation resulted in one peptide of Env, three of Gag and 12 of Pol. Only Gag340-348, Pol83-92, Pol267-277 and Pol960-968 showed binding properties to T2 comparable with those of known CTL epitopes Gag76-84 SLYNTVATL and Pol468-476 ILKEPVHGV. A successful primary MHC class I-restricted CTL response was induced against Pol468-476 and Pol267-277 VLDVGDAYFSV, a peptide in a functional sequence of reverse transcriptase (RT). The resulting CD8+ CTL clones were peptide-specific and able to specifically lyse recombinant vaccinia-HIV-1 RT-infected HLA-A*0201-matched B-LCL.The method used to screen proteins sequences for potential CTL epitopes, test selected peptides for binding to MHC class I and induction of an in vitro primary response against optimal binding peptides resulted in the identification of at least one novel conserved CTL epitope. The novel epitope is located in an area crucial for RT activity. This study demonstrates the feasibility of identifying highly conserved HIV-1-derived peptides capable of eliciting novel anti-HIV-1 CTL responses.

We previously described a motif prediction of major histocompatibility complex allele-specific peptides and an in vitro assay for actual measurement of peptide binding to human leukocyte antigen HLA-A2.1 molecules. Using this method we have identified candidate cytotoxic T lymphocyte (CTL) epitopes derived from a non-self-protein (influenza matrix) and self-protein (p53). We now show that results of binding assays performed over a range of peptide concentrations indicate that distinct differences in HLA-A2.1 peptide binding affinities exist between the influenza matrix and p53 protein. The results for the influenza matrix protein indicate that the peptide that shows the highest binding affinity to HLA-A2.1 is identical to the known immunodominant peptide recognized by influenza virus-specific CTLs. The results for p53 indicate that one of the peptides with a low binding affinity is capable of inducing specific CTL responses, but CTLs recognizing the highest affinity binding peptides were not obtained. These findings are discussed in terms of the distinct implications for induction of cellular immune responses directed against peptides with different binding affinities for HLA-A2.1 of proteins that constitute attractive targets for tumor immunotherapy.

Chronic serum sickness was induced in four groups of Wistar rats by immunization with BSA, cationized BSA (cBSA), human IgG (HuIgG), or human IgM (HuIgM), followed by repeated intraperitoneal (i.p.) injection of the antigen used, to study the effect of the characteristics of an antigen on renal immunopathology. Renal tissue sampled 2, 4, 7, and 9 weeks after the start of the i.p. injections was examined by light-, immunofluorescence-, and electron-microscopy. Proteinuria was measured in urine collected over 24 h. All animals given BSA, cBSA, or HuIgG developed progressive renal disease characterized by initial deposition of antigen and rat Ig in the mesangium of rats given BSA or HuIgG, and minimal amounts in those given cBSA, followed by the appearance in the first instance of subendothelial deposits in the animals receiving BSA or HuIgG, and later subepithelial deposits in those given BSA, cBSA, or HuIgG. The appearance of immunoglobulin deposits along the glomerular capillary wall was associated with the onset of massive proteinuria reaching average levels of 450 mg/24 h for rats given BSA or cBSA, and 500 mg/24 h for those given HuIgG. Animals injected with HuIgM showed only mesangial deposits of human IgM and rat Ig without the development of proteinuria. Under light-microscopy, rats given BSA, cBSA, or HuIgM showed minimal abnormalities, whereas those receiving HuIgG showed transient but severe influx of granulocytes in glomeruli with the development of diffuse proliferative glomerulonephritis in association with a long-lasting phase characterized by subendothelially localized immune aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)