Coculture Of Peripheral Blood Lymphocytes Research Articles

Generation of murine tumour-reactive T cells by co-culturing murine pancreatic cancer organoids and peripheral blood lymphocytes

Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at a late stage and becomes resistant to several treatments. Significant clinical effects have been reported for cancer immunotherapies on a subset of patients diagnosed with epithelial cancers. Cancer organoid co-culture with autologous peripheral blood lymphocytes offers an innovative immunotherapeutic approach that is increasingly being tested, although there is a lack of cutting-edge platforms enabling the investigation of cancer-T cell interactions for individual patients. In this study, a pancreatic cancer organoid culture from a genetically engineered pancreatic cancer murine model was established and co-cultured with autologous peripheral blood lymphocytes to induce a tumour-specific T cell response to pancreatic cancer. Co-culturing autologous peripheral blood lymphocytes with cancer organoids can be an effective strategy to enrich tumour-reactive T cells from the peripheral blood of murine models; this approach could potentially be transferred to humans. Co-culture of peripheral blood lymphocytes and cancer organoids could provide an unbiased approach to evaluating the sensitivity of tumour cells to T cell-mediated priming on an individual patient level.

IL-10 Restores MHC Class I Expression and Interferes With Immunity in Papillary Thyroid Cancer With Hashimoto Thyroiditis.

The incidence of papillary thyroid cancer (PTC) with concomitant Hashimoto thyroiditis (HT) is increasing. Interleukin (IL)-10 is a cytokine previously reported to be elevated in this condition. Evidence from multiple human malignancies showed IL-10 participated in tumor immunity and exhibited therapeutic potential. The aim of this study is to investigate whether IL-10 interferes with tumor immunity in PTC with concomitant HT. Expression of IL-10 and major histocompatibility complex (MHC) class Ⅰ were compared with PTC tissues with or without concomitant HT. PTC cell lines K1 and TPC-1 were stimulated with IL-10 and analyzed for MHC class Ⅰ expression afterward. T-cell activation, production of IL-2 and interferon (IFN)-γ and programmed death-1 (PD-1) expression were assessed by coculture of donor peripheral blood lymphocytes (PBLs) with IL-10-pretreated PTC cells. Programmed death-ligand 1 (PD-L1) expression was measured in PTC tissues and IL-10-pretreated cells of K1 and TPC-1. Increased levels of IL-10 and MHC class Ⅰ were observed in PTC with concomitant HT. IL-10 stimulation increased MHC class Ⅰ expression of PTC cells in vitro. Coculture of PBLs with IL-10-pretreated PTC cells enhanced T-cell activation (% cluster of differentiation [CD]25+ of CD3+T cells) and increased IL-2 production along with decreased IFN-γ secretion and PD-1 expression. Reduced PD-L1 expression was seen in PTC + HT tissue samples and IL-10-stimulated PTC cell lines. Elevated IL-10 expression in PTC with concomitant HT restores MHC class Ⅰ expression and interferes with tumor immunity. The potential mechanism of IL-10 in tumor immunity needs further investigation.

IL-17A increases MHC class I expression and promotes T cell activation in papillary thyroid cancer patients with coexistent Hashimoto\u2019s thyroiditis

BackgroundThe incidence of coexisting papillary thyroid cancer (PTC) and Hashimoto’s thyroiditis (HT) is increasing. The impact of HT on PTC prognosis and its possible mechanism remains controversial. Interleukin-17A (IL-17A) has been reported to participate in the pathogenesis of multiple autoimmune diseases and cancers. The aim of this study is to investigate the role of IL-17A in PTC with coexistent HT and evaluate the changes in tumor antigenicity.MethodsExpression of IL-17A and major histocompatibility complex (MHC) class I molecules were compared on PTC tissue samples with or without HT. PTC cell lines K1 and TPC-1 were stimulated with IL-17A and analyzed for MHC class I expression afterwards. Cluster of differentiation (CD) 8+T cell activation, production of Interleukin-2 (IL-2) and Interferon-gamma (IFN-γ) as well as the programmed death-1 (PD-1) expression on lymphocytes were assessed by coculture of donor peripheral blood lymphocytes (PBLs) with IL-17A pretreated PTC cells.ResultsElevated IL-17A and MHC class I expression were observed in PTC tissue samples with coexistent HT. Stimulation of PTC cells with IL-17A effectively increased MHC class I expression in vitro. Coculture of PBLs with IL-17A pretreated PTC cells resulted in enhanced T cell activation (%CD25+ of CD3+T cells) and increased IL-2 production along with decreased IFN-γ secretion and PD-1 expression of the lymphocytes.ConclusionsPapillary thyroid cancer with coexisting Hashimoto’s thyroiditis presents elevated MHC class I expression, which may be the result of IL-17A secretion. T cell activation is enhanced in vitro by IL-17A and may provide future utility in PTC immunotherapy.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Generation of Tumor-Reactive T Cells by Co-culture of Peripheral Blood Lymphocytes and Tumor Organoids

Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer T-cell interactions for individual patients and understand determinants of responsiveness are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific Tcell responses to epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich tumor-reactive Tcells from peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. Furthermore, we demonstrate that these Tcells can be used to assess the efficiency of killing of matched tumor organoids. This platform provides an unbiased strategy for the isolation of tumor-reactive Tcells and provides a means by which to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient.

BET Bromodomain Inhibition Selectively Targets the Epstein-Barr Virus Oncogene LMP1 While Promoting Virus-Specific, Adaptive Anti-Tumor Activity

Epstein-Barr virus (EBV) is a human herpesvirus that infects over 90% of the world's population and is associated with a wide-range of diseases. EBV has evolved to manipulate host cellular networks in the absence of proper immune function and is linked to malignant lymphoproliferative disorders (LPD) such as Hodgkin's lymphoma and diffuse large B-cell lymphomas. The lack of standard or effective therapeutic approaches for individuals with these aggressive and clinically complicated diseases represents an important unmet need.We have recently demonstrated EBV-induced B cell transformation to be dependent upon the dysregulation of protein arginine methyltransferase 5 (PRMT5), an epigenetic writer that functions as a global transcriptional repressor. We hypothesize that epigenetic readers, writers and erasers are similarly dysregulated following EBV infection of B cells and that these enzymes represent novel therapeutic candidates. The bromodomain and extra-terminal (BET) protein bromodomain 4 (BRD4) is an epigenetic reader that binds acetylated lysine residues and promotes transcription of genes that drive cell growth and survival. Here, we evaluate the relevance of BRD4 to EBV-driven B cell transformation and identify it as a potential therapeutic target for EBV-LPD. We used the highly selective and potent BRD4 inhibitor (BRD4i), JQ1, as a model to examine the relevance of BRD4 in EBV-driven lymphoproliferative disease (LPD).JQ1 treatment of, EBV-transformed lymphoblastoid cell lines (LCLs) led to reduced proliferation, but not direct cytotoxicity. However, similar concentrations of JQ1 in a more physiologically-relevant co-culture setting of EBV-LCLs cultured with autologous peripheral blood lymphocytes (PBMC), resulted in a robust depletion of LCLs, loss of suppressive myeloid populations (TAM/M2 phenotype) and expansion of anti-tumor adaptive, memory CD3/CD8 immune effector cells. Using a single dose of JQ1 (500nM) in a co-culture of LCLs and autologous PBMCs, there was a marked expansion of activated, effector memory (CD3+, CD8+, CD45RO+, CD62L-) cytotoxic T-cell population as compared to vehicle-treated co-cultures. Furthermore, a dramatic reduction in both CD19+ LCLs and suppressive myeloid populations (CD33+, CD11b, HLADR+, CD206+, PD-L1+) was observed after 10 days. Despite the depletion of myeloid cell subsets by CD33 depletion, we still saw the outgrowth of cytotoxic T cell population in the presence of JQ1.In order to delve into the causes of this heightened immune response, we looked at the changes in the expression of highly immunogenic EBV specific proteins. BRD4 inhibition led to upregulation of the EBV oncoprotein, latent membrane protein 1(LMP1), as well as MHC class I antigen presentation machinery in LCLs. The enhanced expression of LMP1 in LCLs with BRD4i, led to modulation of downstream signaling networks driven by this oncogene including PI3K/pAKT (decreased), NF-ĸB (canonical down/non canonical up) and pSTAT3 (decreased). Because LMP1 oncogenic activity is vital for EBV-driven B cell immortalization and transformation, we next conducted in-vitro assays where primary, naïve B cells were infected with EBV in the presence and absence of JQ1 (50, 500nM) treatment. Selective BRD4i was introduced in separate transformation cultures in seven-day intervals following EBV infection and absolute cell counts were monitored. At all timepoints BRD4i prevented EBV-driven transformation of purified naïve B-lymphocytes.Our findings highlight the utility of BRD4i as an experimental therapeutic strategy for EBV-driven lymphomas as it functions to target pathways initiating and sustaining transformed B cell outgrowth and survival while supporting anti-tumor host memory, immune networks. Collectively, these experiments identify BRD4 as a key driver of cell cycle progression, oncogene activation, and a potential immune checkpoint modulator in transformed B lymphoblasts. Furthermore, BRD4i enhances host immunity by eliminating suppressive myeloid cell populations permitting the activation and expansion of memory CD3+/CD8+ cytotoxic T cells. This work demonstrates BRD4i is an attractive therapeutic strategy as it sensitizes malignant cells while enhancing the responsiveness of the host immune system, making it an ideal candidate to be used in future trials with T cell immune therapies. DisclosuresBaiocchi:Essanex: Research Funding.

PMHC/scFv fusion protein mediates killing of tumor cells by viral specific cytotoxic T cells

Objective To study whether the HBC-A2/scFv fusion protein mediates killing of tumor cells by viral specific cytotoxic T cells. Methods The fusion protein was attached to the CD71-expressing, HLA class Ⅰ negative tumor cells. And then, cytolysis by viral peptide-specific CTLs which were generated by co-culture of peripheral blood lymphocytes from HLA-A2 positive donors with inactivated T2 cells pulsed with the viral peptide were tested by lactate dehydrogenase (LDH) releasing. Results The fusion protein can attach the active viral peptide/HLA-A2 complex to K562, HepG2 and U937 cells through binding of CD71 scFv to CD71 (37.30% ±8.25%, 27.20% ±3.88%, 21.80% ±6.49% ) and mediate cytotoxicity of viral peptide-specific CTLs against those cells in vitro ( K562: 42.08% ± 1.14% vs 8.07%± 1.39%; HepG2: 49.72% ± 1.59% vs 12.46% ± 1.26%; U937: 39.72% ± 3.26% vs 7.13% ±1.48% ). Conclusion This viral peptide/HLA-A2 complex targeted by CD71 scFv is able to redirect viral peptide-specific T-cell mediated immune responses against tumor cells. Key words: Viral peptide/HLA-A2 complex; CD71 single chain antibody; Fusion protein; Tumor cell

Human mesenchymal stem cell co-culture modulates the immunological properties of human intervertebral disc tissue fragments in vitro

The capacity of mesenchymal stem cells (MSCs) to differentiate into intervertebral disc (IVD)-like cells has been well described, but their ability to modulate the inflammatory processes in the IVD remains unclear. We found that tissue obtained by discectomy of degenerated and post-traumatic IVD contains significant amounts of IgG antibodies, a sign of lymphocyte infiltration. Further we investigated whether MSCs in vitro, which were characterized for their multilineage differentiation potential and may have immunomodulatory effects on IVD fragments. IVD fragments were co-cultured in contact with peripheral blood lymphocytes (PBLs) and MSCs, and as functional controls we used contact co-cultures of PBLs stimulated with pokeweed mitogen (2.5 μg/mL) and MSCs. The time course of lymphocyte proliferation (Alamar Blue), IgG (ELISA) and gene expression (RT-PCR) of anti-inflammatory cytokines (TGF-β1, IL-10) by MSCs and pro-inflammatory molecules (IL-1α, IL-1β and TNF-α) by the IVD fragments were analyzed. Depending on the response to the presence of MSCs, the IVD fragments (n = 13) were divided in two groups: responders (n = 9), where inflammation was inhibited by MSCs and non-responders (n = 4), where MSCs did not decrease inflammation. At 1 week in co-culture, MSCs reduced significantly the IgG production in the IVD responders group to 69% and PBLs proliferation to 57% of the control. MSCs expression of the anti-inflammatory TGF-β1 increased with time, while IL-10 was expressed only at day 1. IVD gene expression of TNF-α decreased constantly, whereas IL-1α and IL-1β expression increased. In conclusion, these data suggest that MSCs may modulate disc-specific inflammatory and pain status and aid regeneration of the host tissue.

Viral specific cytotoxic T cells inhibit the growth of TfR-expressing tumor cells with antibody targeted viral peptide/HLA-A2 complex

A fusion protein of single chain antibody (scFv) specific for transferrin receptor (TfR, CD71) and viral peptide/HLA-A2 complex was prepared in this study to redirect cytotoxic T cells (CTLs) of viral specificity to tumor cells by attaching the ligand of T cell receptor (TCR) to tumor cells via binding of TfR scFv to TfR. The results demonstrate that the fusion protein can attach the active virus-peptide/HLA-A2 complex to HLA class I-negative, TfR-expressing K562 cells through binding of TfR scFv to TfR, and mediate cytotoxicity of viral peptide-specific CTLs against K562 cells in vitro. In addition, the fusion protein can induce inhibition of solid tumor formation and improve survival time in tumor xenograft nude mouse with the injection of the sorted viral peptide-specific CTLs generated by co-culture of peripheral blood lymphocytes from HLA-A2 positive donors with inactivated T2 cells pulsed with the viral peptide.

Distribution of Several Activating and Inhibitory Receptors on CD3−CD16+ NK Cells and Their Correlation with NK Cell Function in Healthy Individuals

The aim of this study was to estimate the distribution and density of a representative set of activating and inhibitory receptors on gated natural killer (NK) cells, as well as on their bright and dim subsets, and to correlate the receptor expression with NK cell activity for healthy individuals on CD3(-)CD16(+) NK cells. We show that in 43 healthy controls NK cell activity against K562 target cells was 37.34% (E:T, 80:1) by standard chromium release assay. The expression of receptors on NK cells and their subsets was analyzed by flow cytometry. The cytotoxic CD3(-)CD16(bright) NK subset constituted 78.97%, while the regulatory CD3(-)CD16(dim) NK subset constituted 21.03% of NK cells. We show the distribution of NKG2D, CD161, CD158a, and CD158b receptors on CD3(-)CD16(+) NK cells in peripheral blood lymphocytes (PBLs), on gated NK cells, and on the CD3(-)CD16(bright) and CD3(-)CD16(dim) subsets. Contrary to CD158a and CD158b killer immunoglobulin-like receptors (KIRs), there is a significant positive correlation of NKG2D and CD161 expression with NK cytotoxicity. We show the kinetics of change in CD3(-)CD16(+)NK/K562 conjugate composition, together with the stronger target binding capacity of CD16(bright) NK cells. Furthermore, we show that after coculture of PBLs with K562 the expression of CD107a, a degranulation marker, on CD3(-)CD16(+)NK cells and subsets is time dependent and significantly higher on the cytotoxic CD3(-)CD16(bright) NK subset. The novel data obtained regarding expression of NK cell activating and inhibitory receptors for healthy individuals may aid in detecting changes that are associated with various diseases.

Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo.

Human glioblastoma is a highly lethal tumor known for its capability of interfering with effective antitumor immune responses. Costimulatory signals are of critical relevance in both the inductive and effector phases of immune responses. Inducible costimulator-ligand (ICOSL), a member of the B7 family of costimulatory molecules related to CD80/CD86, regulates CD4 as well as CD8 T-cell responses via interaction with its receptor, ICOS, on activated T cells. We report the expression of ICOSL by glioma cells in vitro and in vivo. In contrast to CD80 (B7.1) and CD86 (B7.2), ICOSL protein and mRNA was expressed in 7 of 12 glioma cell lines. ICOSL expression is upregulated by the inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), whereas interferon-gamma (IFN-gamma) has no such effect. Further, immunohistochemical analysis of human brain tumors demonstrates the expression of ICOSL in three of four tissue samples. ICOSL expression is functional in that a neutralizing ICOSL antibody (HIL-131) reduces Th1 and Th2 cytokine levels in cocultures of peripheral blood lymphocytes or T-cell subsets (CD4 and CD8) with glioma cells. However, ICOSL gene transfer into glioma cells does not alter their immunogenicity under primary or secondary alloreactive coculture assays.

Spontaneous and anti-Fas antibody-mediated apoptosis of the peripheral blood lymphocytes in Sjögren’s syndrome, rheumatoid arthritis and systemic lupus erythematosus

The objective of this study was to clarify possible roles of lymphocyte apoptosis in autoimmune rheumatic diseases. Spontaneous and anti-Fas antibody-mediated apoptosis of peripheral blood (PB) lymphocytes were measured by a flow cytometric method using propidium iodide in primary Sjogren’s syndrome (SS), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Spontaneous apoptosis levels were significantly higher in 26 SLE patients than in 19 healthy controls. The apoptosis levels were not significantly different between 19 primary SS patients or 28 RA patients and the controls, but high apoptosis levels were observed in some of the patients. The apoptosis of PB lymphocytes was also enhanced in seven SS patients with RA and 16 SS patients with SLE. Anti-Fas antibody could induce apoptosis of PB lymphocytes from both healthy controls and patients’ groups. The antibody-mediated apoptosis levels were higher in RA, SLE and secondary SS patients with RA or SLE, but not in primary SS patients. Each patients’ group was further divided into two groups according to their mean apoptosis levels: patients with high and those with low spontaneous or antibody-mediated apoptosis levels. Clinical variables reflecting disease activity for each disease were then compared between the two groups. Serum rheumatoid factor (RF) and anti-SS-A/Ro antibody titers were higher in the RA or primary SS patients with high spontaneous apoptosis levels than those with low apoptosis levels, respectively. When all the patients’ groups were evaluated together, the spontaneous apoptosis levels negatively correlated with PB lymphocyte counts. In addition, the spontaneous apoptosis levels were decreased by the co-culture of PB lymphocytes with interleukin-2 (IL-2: 100 U/ml) in most individuals including patients and healthy controls. We conclude that spontaneous apoptosis of PB lymphocytes was enhanced in SLE and secondary SS patients. Production of RF or anti-SS-A/Ro antibodies was associated with enhanced apoptosis of PB lymphocytes in RA or primary SS patients. The apoptosis levels were related with lymphocytopenia observed in the patients examined, although various factors including IL-2 seemed to be protective against the apoptosis of circulating lymphocytes. Furthermore, anti-Fas antibody induced apoptosis of PB lymphocytes from the healthy controls and patients, and the antibody-mediated apoptosis levels were high in RA, SLE and secondary SS patients.

Spontaneous and anti-Fas antibody-mediated apoptosis of the peripheral blood lymphocytes in Sjögren’s syndrome, rheumatoid arthritis and systemic lupus erythematosus

The objective of this study was to clarify possible roles of lymphocyte apoptosis in autoimmune rheumatic diseases. Spontaneous and anti-Fas antibody-mediated apoptosis of peripheral blood (PB) lymphocytes were measured by a flow cytometric method using propidium iodide in primary Sjögren’s syndrome (SS), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Spontaneous apoptosis levels were significantly higher in 26 SLE patients than in 19 healthy controls. The apoptosis levels were not significantly different between 19 primary SS patients or 28 RA patients and the controls, but high apoptosis levels were observed in some of the patients. The apoptosis of PB lymphocytes was also enhanced in seven SS patients with RA and 16 SS patients with SLE. Anti-Fas antibody could induce apoptosis of PB lymphocytes from both healthy controls and patients’ groups. The antibody-mediated apoptosis levels were higher in RA, SLE and secondary SS patients with RA or SLE, but not in primary SS patients. Each patients’ group was further divided into two groups according to their mean apoptosis levels: patients with high and those with low spontaneous or antibody-mediated apoptosis levels. Clinical variables reflecting disease activity for each disease were then compared between the two groups. Serum rheumatoid factor (RF) and anti-SS-A/Ro antibody titers were higher in the RA or primary SS patients with high spontaneous apoptosis levels than those with low apoptosis levels, respectively. When all the patients’ groups were evaluated together, the spontaneous apoptosis levels negatively correlated with PB lymphocyte counts. In addition, the spontaneous apoptosis levels were decreased by the co-culture of PB lymphocytes with interleukin-2 (IL-2: 100 U/ml) in most individuals including patients and healthy controls. We conclude that spontaneous apoptosis of PB lymphocytes was enhanced in SLE and secondary SS patients. Production of RF or anti-SS-A/Ro antibodies was associated with enhanced apoptosis of PB lymphocytes in RA or primary SS patients. The apoptosis levels were related with lymphocytopenia observed in the patients examined, although various factors including IL-2 seemed to be protective against the apoptosis of circulating lymphocytes. Furthermore, anti-Fas antibody induced apoptosis of PB lymphocytes from the healthy controls and patients, and the antibody-mediated apoptosis levels were high in RA, SLE and secondary SS patients.

Expression of CD80 Enhances Immunogenicity of Cervical Carcinoma Cellsin Vitro

Although cervical carcinoma cells may express the human papillomavirus oncoproteins E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. This failure may be due to a lack of expression of costimulatory molecules, such as CD80 (B7.1). To augment the immunogenicity of cervical carcinoma cells, we transfected human papillomavirus (HPV)-transformed cell lines, CaSki and HeLa, with the CD80 expression vector pBJ. Alloantigens on the tumor cells were used for the stimulation of peripheral blood lymphocytes (PBLs). Cocultivation of PBLs and tumor cells resulted in proliferation of CD4+and CD8+T lymphocyte subsets. CD80-expressing tumor cells induced proliferation of allogeneic PBLs two- to sixfold compared to control cell lines. Cocultivation of allogeneic PBLs with CD80-positive tumor cells for 3 weeks gave rise to cytotoxic T cells capable of lysing untransfected parental tumor cell lines. Our results demonstrate an immunostimulatory effect of CD80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.

Increase in Sensitivity to Soluble CD4 by Primary HIV Type 1 Isolates after Passage through C8166 Cells: Association with Sequence Differences in the First Constant (C1) Region of Glycoprotein 120

Primary isolates of human immunodeficiency virus type 1 (HIV-1) were obtained by coculture of peripheral blood lymphocytes (PBLs) from HIV-1-infected people with PBLs from uninfected donors. These viral stocks tend to be resistant to neutralization/inactivation by soluble CD4 (sCD4). When these stocks were passed through the T cell line C8166, virus stocks emerged that were sensitive to sCD4. Pre- and post-C8166 stocks maintained their sCD4-resistant and -sensitive phenotypes, respectively, with further passage in PBLs. Pre- and post-C8166 stocks were biologically cloned by two cycles of limiting dilution. The majority (14 of 17) of pre-C8166 clones were sCD4 resistant, and, conversely, the majority of post-C8166 clones (11 of 12) were sensitive to sCD4. Nucleotide and predicted amino acid sequence analysis in the env (gp120) region revealed a limited number of differences between the clones. The only differences that sorted with biological phenotype were in the first constant (C1) region of gp120. Adaptation to growth in C8166 cells and conversion from the sCD4-resistant to the sCD4-sensitive phenotype represent the emergence to prominence of viral species in the pre-C8166 stock that have a replication advantage in C8166 coincident with increased sensitivity to sCD4.

T Cell Clones Generated from Patients with Type 1 Diabetes Using Interleukin-2 Proliferate to Human Islet Antigens

T lymphocytes are implicated in the pathogenesis of Type 1 (insulin dependent) diabetes. Activated T lymphocytes expressing IL-2 receptors are found at increased levels in the peripheral blood in the prediabetic period, at diagnosis and for several months after the onset of the disease, but their role in the pathogenesis of the disease is not known. We have used co-culture of peripheral blood lymphocytes with IL-2 alone to selectively generate T cell clones from the in vivo activated T cell population, and examined the phenotype and antigen specificity of the clones derived. From 3 patients with newly-diagnosed Type 1 diabetes 184 clones were generated, the majority of which (39%) were CD4+TCR alpha beta+, whilst 31% were CD8+TCR alpha beta+. From 2 healthy control subjects 90 clones were obtained, of which 62% were CD4+TCR alpha beta+ and 33% were CD8+TCR alpha beta+. Antigen specificity was examined in 46 clones from the patients and 44 from the control subjects in proliferation assays, using as antigens homogenate of human islets of Langerhans, human islet membrane preparation and human liver membrane preparation. Three clones (all CD4+TCR alpha beta+) from the patients, but none from the control subjects, proliferated in a dose dependent fashion in response to stimulation with human islet homogenate presented by autologous APCs, but to neither of the other autoantigen preparations. Our results demonstrate that a relatively high proportion (7%) of T lymphocytes activated in vivo recognise human islet antigens, indicating that they may have a role in the pathogenesis of the disease.

Sequence Analysis of the V3 Loop Regions of theenvGenes of Ugandan Human Immunodeficiency Proviruses

Ugandan strains of human immunodeficiency virus type 1 (HIV-1) were isolated by cocultivation of peripheral blood lymphocytes from infected individuals with cord blood lymphocytes. Sequences from the V3 region of the env gene were amplified by the polymerase chain reaction (PCR) from chromosomal DNA obtained from low passage virus cultures. The PCR products from 13 Ugandan isolates were cloned into a phagemid vector and sequenced. Many isolates contained divergent V3 loop sequences and adjacent regions: diversity was associated with codon deletions or duplications and with nucleotide substitutions, especially G----A transitions. Proviruses from some of the cultures showed extensive diversity within the V3 loop sequences but others were more homogeneous. The V3 loop apices were conserved in 6 of the Ugandan proviruses and these were very similar to the equivalent regions of several Zairean proviruses. The V3 loop apices of African isolates of HIV-1 are divergent from those of North American isolates. The possible biological consequences of this divergence are discussed.

Suppression of lymphokine‐activated killer cell cytotoxicity by a soluble factor produced by squamous tumors of the head and neck

Incubation of human peripheral blood lymphocytes (PBL) in the presence of interleukin-2 results in the generation of lymphokine-activated killer (LAK) cells that are highly cytotoxic to a variety of autologous and allogenic tumor targets. We have identified a noncytotoxic, soluble factor, produced by human squamous cell cancers of the head and neck, that profoundly inhibits the generation of LAK cytotoxicity. Inhibition of the generation of cytotoxicity was demonstrated with coculture of PBL and freshly disaggregated tumor cells in a Transwell two-chamber system. Alternatively, inhibition occurred when LAK cells were generated in the presence of tumor-conditioned supernatants alone. These effects were not observed with conditioned supernatants from autologous or allogenic lymphocytes, human fibroblasts, or the erythroleukemia cell line K562. The presence of this inhibitory factor(s) was not required during the entire period of LAK generation. Suppression of cytotoxicity, measured after 4 days of LAK generation, could also be demonstrated when the conditioned tumor supernatant was present in only the last 24 hours of incubation. Suppression is mediated by a heat-labile factor with a molecular weight of greater than 75 kd. These results suggest that LAK cytotoxicity may be significantly impaired by soluble immunoregulatory factors present within the tumor milieu of squamous cell carcinomas of the head and neck. Further characterization of these factors may lead to the development of more rational and effective forms of immunotherapy.

Human suppressor T cells induced in vitro with an autologous renal allograft-derived T cell line I. Suppressor cell induction, function, and specificity

The functional characteristics of T suppressor (Ts) cells generated from the peripheral blood lymphocytes (PBL) of a kidney transplant recipient who had excellent graft function for 1 year were examined. Ts cells were induced by co-culture of PBL with an autologous alloreactive cytotoxic T lymphocyte (CTL) line (EE-1) previously grown from a routine renal allograft biopsy of this patient performed 10 days posttransplant. The EE-1 line included CD3 + T cells of CD8 + and CD4 + phenotypes with cytotoxic specificity for disparate class I (HLA-B8) and class II (HLA-DR1 and 3) antigens of the kidney donor (JC). The EE-1 induced Ts cell lines (designated TsEE) were found to significantly suppress (50%–95%) autologous fresh responder EE-PBL stimulation by donor EBV-transformed cells (JC-EBV) in mixed lymphocyte reaction (MLR) assay. TsEE cells were CD3 + (98%) and predominantly CD8 + (68–80%), showed no cytotoxic activity, and were suppressive only at the early phase of MLR stimulation. In three-party cell test MLR assays, TsEE-mediated suppression appeared restricted to responder cells sharing HLA-B7 with the suppressor line, and was not abrogated by the addition of exogenous interleukin-2 (IL-2). TsEE cells also showed restricted suppression of CTL generation but not mature CTL activity. The restricted suppressor activity of TsEE lines was dependent upon their induction and restimulation with the autologous EE-1 line.

Utilization of tumour-sensitized ('educated') and radiolabelled lymphocytes for tumour localization.

Upon coculture of peripheral blood lymphocytes (PBL) in the presence of irradiated autologous tumour cells, the PBL can be sensitized to the tumour (or perhaps more correctly resensitized), as shown by in vitro cytotoxic properties. The cells can be proliferated in the presence of interleukin-2, radiolabelled with 111In and injected back into the cell donor. Using this technique, tumour deposits were localized in five out of seven patients. Possible techniques for increasing the specificity of this form of adoptive immunocytotherapy are pointed out.