Colonic mucosal T lymphocytes in ulcerative colitis: Expression of CD7 antigen in relation to MHC class II (HLA-D) antigens

T-cell subsets and their activation state were examined by double-label immunofluorescence of cryostat tissue sections of the colon from 21 patients with ulcerative colitis (UC) and 30 histologically normal controls. Expression of MHC class I (HLA-A, B, C) and class II (HLA-D) antigens was studied in parallel. In the normal colonic mucosa, the CD4CD8 ratio in the epithelial compartment approximated 11, and in the lamina propria, 2.551. Of the CD8⁺ (cytotoxic/suppressor) subset, approximately half did not express the CD5 pan-T marker in either compartment. Virtually no Leu 8⁺ cells were observed, implying that the CD4⁺ subset consisted of helper, rather than suppressor-inducer cells. Classical markers of T-cell activation (CD25, HLA-D) and proliferation were absent, and strong expression of the CD7 immunostimulation marker was approximately equal in both CD4 and CD8 subsets. The epithelium was uniformly negative for class II antigens, but positive for class I. In UC, there were no significant alterations in CD4CD8 ratios in either compartment, and there were no changes with respect to phenotype of the subsets. In 11 of 19 patients (mainly with total colitis), enterocytes were HLA-D⁺. In this HLA-D⁺ group, there was an increase in the percentage of CD4⁺ cells coexpressing CD7; this difference was significant (P<0.02) in the lamina propria. Increased expression of CD7 was also found by the CD6⁺ T cell subset (P<0.05). These results suggest that class II expression is mediated by immunostimulated T helper cells in UC, with consequences for antigen presentation and maintenance of the chronic inflammatory state.HLA-D is used as a generic term for class II major histocompatibility complex (MHC) gene products (HLA-DR, DP, DQ) unless specified otherwise.     

Phenotypic and functional characterization of T-cell lines generated from colonoscopic biopsy specimens in patients with ulcerative colitis

Intestinal T-cell lines were generated from lamina propria mononuclear cells isolated from colonoscopic biopsies in ulcerative colitis patients and controls. In both ulcerative colitis and controls, expanded cells were constituted largely by T-cell receptor ⁺, CD4⁺, CD45RA^– (helper), and CD8⁺, CD11b^– (cytotoxic) phenotypes. T-cell receptor V gene usage was not significantly changed after cell expansion and no difference was observed between ulcerative colitis and controls. Ulcerative colitis cells, especially those derived from the patients with long-standing disease, showed significantly higher levels of cytotoxicity against the target cells, including those of colonic epithelial origin, and enhanced production of tumor necrosis factor- and interferon- after short incubation with anti-CD3 antibody. Generation of T-cell lines from colonoscopic biopsy specimens may be useful for detailed functional characterization of locally infiltrating T cells in ulcerative colitis patients.     

Intraepithelial lymphocytes in colon have similar properties to intraepithelial lymphocytes in small intestine and hepatic intermediate TCR cells

Recently, properties of intraepithelial lymphocytes (IEL) in the colon (C-IEL) have been analyzed in comparison with those of IEL in the small intestine (SI-IEL). We compared the properties of C-IEL with those of SI-IEL and hepatic intermediate TCR cells, two other types of extrathymic T cells. C-IEL and intermediate TCR cells contain many NK1⁺T cells, although SI-IEL contain few. V and V usage of C-IEL was the same as that of SI-IEL, and that of intermediate TCR cells was different. C-IEL responded to Con A while SI-IEL did not. As to adhesion molecules, C-IEL include both extrathymic and thymus-originated type T cells. With age, TCR-⁺CD4⁺CD8⁺ cells do not increase among C-IEL but do increase among SI-IEL. IL-2R⁺ or CD4^–CD8^– C-IEL increase as observed in the liver. These results indicate that these organ-specific T cells have different roles at their respective sites and that they may be of different lineages.     

Specific CTL Activity of CD8+ TCR Vβ14+ T Cell in Mouse 2, 4, 6-Trinitrobenzene Sulfonic Acid-Induced Colitis

We analyzed the functional role of CD8⁺ T-cell receptor (TCR) V14⁺ T cells, which increased specifically in the lamina propria in 2,4,6-trinitrobenzene sulfonic acid (TNBS) -induced colitis. Cytotoxic activity and cytokine production in CD8⁺ TCR V14⁺ T-cell clones were analyzed by ⁵¹Cr release assay and enzyme-linked immunosorbent assay, respectively. Cell transfer studies using these clones were performed. Established T-cell clones showed specific cytotoxic activity against TNBS-conjugated self spleen cells, and this cytotoxicity was completely inhibited by anti-TCR V14 monoclonal antibody. These clones produced interferon (IFN) - in their culture supernatant, but neither interleukin (IL) - 2 nor IL-4. Histological findings of the colon in mice, which received clone transfer after enema with suboptimal doses of TNBS, showed massive colitis. Our results indicate that CD8⁺ TCR V14⁺ T cells had a cytotoxic T-lymphocyte function induced by Th-1 T-cell response and played a pathogenic role in the development of TNBS-induced colitis.     

TCR1+ large granular lymphocyte proliferation in rheumatoid arthritis

The T-lymphoproliferative syndrome is characterized by a proliferation of large granular lymphocytes (LGL). It is often associated with neutropenia, and in 30% of cases with rheumatoid arthritis (RA). Phenotypic analysis has demonstrated that in most cases of RA with T-proliferative disease, the LGL represent T cells with a clonal rearrangement of the / T cell receptor (TCR2). Here, three patients with / TCR1⁺ LGL proliferation suffering from long-standing arthritis and neutropenia are described. The first patient with RA showed an expansion of a heterogeneous CD2⁺ CD16⁺ CD56^- LGL population, of which 30% coexpressed TCR1 with V1 rearrangement. The second patient with ankylosing spondylitis and RA was suffering from proliferation of TCR1⁺ (V9^-, V1^-), CD2⁺ CD16^- CD56^- LGL with low coexpression of CD8. The third patient with RA was suffering from a proliferation of TCR1⁺ (V1⁺, V9^-) CD4^- CE8^- CD16^- CD56^- lymphocytes. On the basis of these unusual findings, the pathogenetic role of TCR1⁺ T cells in RA is discussed.     

γ-Glutamyl transpeptidase-cellular expression in populations of normal human mononuclear cells and patients suffering from leukemias

The expression of the ectoenzyme-glutamyl transpeptidase (EC2.3.2.2.,GT) was investigated by flow cytometry on populations of peripheral blood mononuclear cells (PBMC) from healthy subjects and patients suffering from several types of leukemia before and under chemotherapy. In unstimulated PBMC, 28% of these cells were found to beGT positive. The highest expression was measured on monocytes (CD14/GT⁺ cells: 60%). Within the subsets of T lymphocytes (CD3/GT⁺ cells: 18%) we saw no clear differences between CD4⁺ and CD8⁺ cells. B lymphocytes, NK cells, and activated cells showed low expressions (up to 10%). Treatment of PBMC with mitogens, -IFN, IL-2, and GM-CSF did not affect the enzyme expression on normal mononuclear cells (MNC). However, a rapid increase of yGT⁺ cells was found in the presence of glutathione (GSH) and n-acetyl cysteine (nAC), particularly on monocytes, B cells, and NK cells. Comparing 40 healthy subjects and untreated patients suffering from leukemias, a significantly higher expression ofGT⁺ cells in the total MNC populations (B-CLL: 57%, CML: 62%GT⁺ cells) was observed in B-chronic lymphocytic leukemia (B-CLL) and chronic myelogenous leukemia (CML), whereas other leukemias did not show clear differences. Most interestingly, theGT expression was diminished in all populations of CML cells after 5 h of incubation in the presence of 10 units/ml IFN-. These data suggest a possible protective role ofGT in MNC and a regulatory function of this enzyme in the development of CML.     

Suppression of diabetes mellitus in the non-obese diabetic (NOD) mouse by an autoreactive (anti-I-Ag7) islet-derived CD4+ T-cell line

Summary The non-obese diabetic (NOD) mouse is a spontaneous model of human insulin-dependent diabetes mellitus. Both CD4⁺ and CD8⁺ T cells infiltrate the pancreatic islets of NOD mice prior to beta-cell destruction. T-cell lines isolated from the islets of NOD mice are tools for studying the pathogenesis of insulin-dependent diabetes mellitus. During attempts to generate such lines we isolated an autoreactive CD4⁺ T-cell line, designated C2, from the insulitis lesion of a 20-week-old female non-diabetic NOD/WEHI mouse. Islet T cells were propagated by the addition of interleukin-2 and reexposure every 2 weeks to whole NOD islets and irradiated NOD spleen cells as antigen presenting cells. C2 cells proliferated up to 100-fold upon exposure to NOD antigen presenting cells but did not respond to whole NOD islets or antigen presenting cells from allogeneic mouse strains. Proliferation of C2 cells to NOD antigen presenting cells was blocked by a monoclonal antibody against the unique class II MHC molecule of NOD, I-A_g7. In response to NOD antigen presenting cells, C2 cells secreted Interferon-, tumour necrosis factor- and interleukin-6 but no detectable interleukin-2, interleukin-4 or interleukin-10, a pattern of cytokine secretion more characteristic of Th₁ CD4 cells. C2 cells displayed significant cytotoxicity in a redirected lysis assay. To explore a possible role for autoreactive T cells in the pathogenesis of autoimmune diabetes, C2 cells were injected i.v. into female NOD mice that had received cyclophosphamide to accelerate development of diabetes. Although not different at day 10, by day 16 the frequency of diabetes (blood glucose>15 mmol/l) in mice given cyclophosphamide and C2 cells was significantly less than in mice given cyclophosphamide alone (p<0.05). These results are consistent with the suppressor function of autoreactive T-cells previously reported and with the concept that autoreactive T cells modulate autoimmune beta-cell destruction in the NOD mouse.     

CD8+CD122+CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity

The Fas/FasL (CD95/CD178) system is required for immune regulation; however, it is unclear in which cells, when, and where Fas/FasL molecules act in the immune system. We found that CD8⁺CD122⁺ cells, which are mostly composed of memory T cells in comparison with naïve cells in the CD8⁺CD122⁻ population, were previously shown to include cells with regulatory activity and could be separated into CD49d^low cells and CD49d^high cells. We established in vitro and in vivo experimental systems to evaluate the regulatory activity of CD122⁺ cells. Regulatory activity was observed in CD8⁺CD122⁺CD49d^low but not in CD8⁺CD122⁺CD49d^high cells, indicating that the regulatory cells in the CD8⁺CD122⁺ population could be narrowed down to CD49d^low cells. CD8⁺CD122⁻ cells taken from lymphoproliferation (lpr) mice were resistant to regulation by normal CD122⁺ Tregs. CD122⁺ Tregs taken from generalized lymphoproliferative disease (gld) mice did not regulate wild-type CD8⁺CD122⁻ cells, indicating that the regulation by CD122⁺ Tregs is Fas/FasL-dependent. CD122⁺ Tregs taken from IL-10–deficient mice could regulate CD8⁺CD122⁻ cells as equally as wild-type CD122⁺ Tregs both in vitro and in vivo. MHC class I-missing T cells were not regulated by CD122⁺ Tregs in vitro. CD122⁺ Tregs also regulated CD4⁺ cells in a Fas/FasL-dependent manner in vitro. These results suggest an essential role of Fas/FasL as a terminal effector of the CD122⁺ Tregs that kill activated T cells to maintain immune homeostasis.Fas (CD95) and its ligand FasL (CD178) compose a unique system that is strongly related to programmed cell death (1). FasL has been considered one of the effector molecules involved in the killing of target cells by cytotoxic T lymphocytes (CTLs) (2). When Fas binds to FasL, it induces downstream signal transduction pathways that subsequently activate cell death induction pathways (3, 4). Thus, the Fas/FasL system appears to act as an effector for CTL-mediated killing of virus-infected or cancer cells, similar to the perforin–granzyme system (5, 6). However, because Fas-mutant (lpr) and FasL-deficient (gld) mice show lymphoproliferative changes, it has been suggested that the Fas/FasL system is important for suppression/regulation of activated effector T cells (7, 8).Molecular mechanisms after Fas activation have been thoroughly investigated, and the signal transduction pathway has been largely elucidated (9, 10). However, research on the cellular events that use the Fas/FasL system has progressed comparatively slowly. There are only a few relevant reports in this respect, mostly of human CD4⁺Foxp3⁺ Tregs that use the Fas/FasL system for their regulatory activity. [In this article, we use the term “Treg(s)” for any kind of T cells that show immune regulatory activity.] To complicate matters further, some contradictory reports claim that such Fas/FasL-engaging Tregs do not exist (8, 11, 12). No reliable reports suggesting that CD8⁺ T cells use the Fas/FasL system for their regulatory action are available. Therefore, it is not clear precisely which subset of T cells express FasL or where Fas/FasL-dependent CD8⁺ Tregs, if such cells exist, are located and at which point they function. Thus, the ultimate pathophysiological role of the Fas/FasL system is still unknown.Regulation of the immune reaction is of pivotal importance for maintaining health in multicellular organisms. In highly developed animals, Tregs, a subset of T lymphocytes especially known as CD4⁺CD25⁺Foxp3⁺ cells, are the main regulators of the immune response (13–16). However, it is not quite clear whether CD8⁺ regulatory T cells exist; there are only few and contradictory reports on their existence, in contrast to the reports on CD4⁺ Tregs. A population of predominantly CD8⁺ suppressor T cells has been described and debated in the 1980s (17, 18). In the 2000s, we found and reported that the cells of central memory phenotype (CD8⁺CD122⁺) also possess the regulatory function, and some other reports regarding CD8⁺ Tregs with some contradictory findings have been published. To date, more than 10 CD8⁺ Treg populations with different markers have been reported (19).One of the best characterized CD8⁺ Treg populations is the CD8⁺CD122⁺ Treg (122⁺ Treg) population. To confirm the existence of CD8⁺ Tregs, markers that may be possible to distinguish Tregs from other T cells were examined. We previously found that CD49d can separate CD8⁺CD122⁺ cells into at least two subsets (CD49d^low and CD49d^high) (20). To judge which cells have a stronger regulatory activity, we prepared two experimental systems: an in vitro system, based on cell culture, and an in vivo system, based on adoptive transfer of T cells.Interestingly, CD8⁺ Tregs express CD122 (IL-2/IL-15 receptor β chain) in contrast to CD4⁺ Tregs, which express CD25 (IL-2 receptor α chain), indicating the fundamental importance of IL-2 at the development/maturation of both CD4⁺ and CD8⁺ Tregs. Suzuki and coworkers generated CD122-deficient mice using gene targeting (21) and used them to identify 122⁺ Tregs (22). In this study, which is a follow-up study to our previous report, we performed in vitro and in vivo experiments to gain further understanding of these cells. We found that CD49d might be a good marker for classifying CD8⁺ T cells into naïve, resting T cells (CD62L⁺CD122⁻), effector memory T cells (CD62L⁻), and T cells of central memory phenotype (CD122⁺CD49d^low), by using multicolor staining of CD62L, CD122, and CD49d. To our knowledge, this is the first report on the role of the Fas/FasL system in the action of CD8⁺CD122⁺ Tregs (122⁺ Tregs) (22–25). Additionally, we show that CD8⁺ Tregs are included in the CD49d^low population, which corresponds to the central memory T-cell population, and that their mechanism of suppression depends on the cytotoxicity mediated by the Fas and FasL interaction.     

T-cell receptor Vβ chain expression in patients with juvenile rheumatoid arthritis

Summary The V chain repertoire in peripheral blood T-cells was analyzed in 23 patients with juvenile rheumatoid arthritis (JRA). Of these, 15 patients had active disease as defined by tender and swollen joints. The ESR was elevated in all but three patients, C-reactive protein (CRP) was elevated in eight. Three patients were investigated during active and inactive phases of the disease. In the active phase of the disease T-cell composition was characterized by an increased number of CD4⁺ helper cells due to a marked increase in the CD4⁺CD45RA⁺ subgroup (34.0±10.9%,P<0.001) and a decrease in CD8⁺CD29⁺ T-cells (10.3±5.6%,P<0.05) compared to controls (15.4±10.0% and 17.8±12.3%, respectively). Using the monoclonal antibodies available to determine T-cell receptor (TCR) expression, patients with active disease demonstrated a significant predominance of T-cells bearing TCRs of the V5 family as determined by flow cytometry (7.6±6.7 vs 3.4±1.3 in controls,P=0.01). In active polyarthritis, up to 24% of peripheral blood T-cells expressed TCRs of the same family. In the majority of JRA patients and especially in non-active disease, no preferential TCRs were found compared to controls. However, even defining only a part of TCRs by immunofluorescence, in certain patients a preferential or dominant expression of a single V gene family in the T-cells was found, supporting the concept of an involvement of T-cells in the autoimmune pathogenesis of JRA.     

Donor lymphocyte infusions for relapse of chronic myeloid leukemia after allogeneic stem cell transplantation: prognostic significance of the dose of CD3+ and CD4+ lymphocytes

Between December 1993 and November 2001, 30 patients with chronic myeloid leukemia who relapsed after stem cell transplantation were studied. Seventeen patients were not treated before donor lymphocyte infusion (DLI), eight patients received interferon- (IFN-), and five underwent chemotherapy. The method of DLI was the bulk dose regimen. The median time between DLIs was 6 weeks. The median number of infusions was three; the median time from transplant to relapse was 17 months and from relapse to DLI 2 months. Eleven patients (37%) were in molecular/cytogenetic relapse, 14 (47%) in chronic phase, and five (16%) in accelerated or blastic phase. Seventeen patients (57%) developed acute graft-versus-host disease (GVHD). Chronic GVHD was observed in 15 of 24 (62%) patients. Four (13%) patients developed cytopenia after a median of 30 days. Nineteen (63%) patients achieved response, 15 of them developed GVHD. The response rate according to the disease phase was molecular or cytogenetic relapse: 91%, chronic phase: 57%, and accelerated or blastic phase: 20%. The median time to response was 6 months. Patients treated with IFN- or no treatment as well as those who were in molecular/cytogenetic relapse and those who received a CD3⁺ cell dose <1×10⁸/kg and CD4⁺ <8×10⁷/kg had better survival. We conclude that patients who receive lower doses of lymphocytes have better survival. In some patients IFN- seems to be a good choice to potentiate the graft-versus-leukemia (GVL) effect.     

Expansion of CD8+CD57+ T cells after allogeneic BMT is related with a low incidence of relapse and with cytomegalovirus infection

Summary. Peripheral blood lymphocytes of 46 recipients of lymphocyte-depleted bone marrow allografts were pheno-typically analysed over a period of 1 year. We investigated the repopulation of lymphocyte subpopulations and their relation with clinical parameters such as graft-versus-host disease (GVHD), graft-versus-leukaemia and cytomegalovirus (CMV) infection. The number of repopulated T cells varied strongly between the blood samples of the recipients. In 45% of the recipients the number of T cells recovered to or above normal levels within 3 months after bone marrow transplantation (BMT), whereas the other recipients remained below normal up to 1 year after BMT. In recipients with a high repopulation, the CD8⁺ T-cell subset contributed more to this high repopulation than the CD4⁺ T-cell subset. We showed that the majority of T cells of these recipients expressed the a/3 T-cell receptor, CD8, CD57 and CDllb. HLA-DR was also highly expressed reflecting the activation stage of T cells in these recipients. BMT recipients with a high repopulation of CD8⁺ T cells showed a lower incidence of leukaemic relapse than recipients with a low repopulation. The 3-year probability of relapse was 19% versus 64% (P=O03), respectively. The relative high number of CD8⁺ T cells at 3 months after BMT was not associated with the incidence of GVHD. In contrast, occurrence of CMV infection after BMT was significantly higher in these recipients. Our results indicate that CD8⁺ T cells, predominantly CD57⁺, of BMT recipients with an expansion of these cells represent an in vivo activated cell population. This CD8⁺ T-cell population may consist partially of cytotoxic cells with anti-leukaemic activity as suggested by a low relapse rate. The signal for the strong expansion of these CD8+CD57+ T cells after BMT is still unclear, but association with CMV infection suggests that viral antigens are involved.     

Decrease in CD4+CD25+ and CD8+CD28+ T cells in interstitial pneumonitis associated with rheumatic disease

Abstract

The expression of CD25 or CD28 on T cells was examined in patients with rheumatic diseases associated with interstitial pneumonitis (IP), in order to investigate the conditions of CD4⁺CD25⁺ regulatory T cells and CD8⁺CD28^? suppressor T cells. Fifty-five patients with various rheumatic diseases and 23 normal controls were enrolled. CD4⁺CD25⁺ T cells of patients with IP were significantly decreased in comparison with non-IP patients, and the ratio of CD8⁺CD28^? T cells in patients with IP was significantly higher than that in non-IP patients or normal controls. These results for CD8⁺CD28^? T cells were in accord with the decrease in CD8⁺CD28⁺ T cells, and may be related to activation-induced CD8⁺CD28⁺ T-cell death. Thus, the abnormality of CD4⁺CD25⁺ regulatory T cells may be related to the pathogenesis of IP, and the survival and activation of CD8⁺ T cells.     

Intratumoral injection of interferon-gamma gene-modified dendritic cells elicits potent antitumor effects: effective induction of tumor-specific CD8<Superscript>+</Superscript> CTL response

Purpose To examine the antitumor efficacy of intratumoral injection of interferon-gamma gene-modified dendritic cells (DC-IFN-) in a B16 melanoma model and to investigate its related immunological mechanisms.Methods C57BL/6 mice-derived DC were transfected with adenovirus encoding IFN- or -galactosidase (DC-LacZ). Secretion of IFN- and TNF- by DC was detected by ELISA. Nitric oxide (NO) production was measured by Griess reaction. Cytotoxicity of DC against tumor cell lines and activity of cytotoxic T lymphocytes (CTLs) were determined by ⁵¹Cr-release assay. TRP-2aa180–188-specific CD8⁺ CTLs in tumor-bearing mice with different treatment were determined by ELISPOT.Results DC-IFN- could secrete high levels of IFN-, NO and TNF-. DC-IFN- were cytolytic to B16 melanoma cells in vitro, but DC-LacZ and DC were not. Significant inhibition of tumor growth and prolonged survival were achieved in tumor-bearing mice intratumorally injected with DC-IFN- when compared with those in tumor-bearing mice intratumorally injected with DC, DC-LacZ, fibroblasts, IFN- gene-modified fibroblasts or PBS. After treatment with DC-IFN-, enhanced Th1 and decreased Th2 responses were observed, and B16 melanoma antigen TRP-2aa180–188-specific CD8⁺ CTLs were induced significantly in the tumor-bearing mice.Conclusions Intratumorally injected DC-IFN- can uptake tumor antigens in situ and cross-present tumor antigens to specific CD8⁺ T cells, hereby eliciting effective antitumor effects in murine model with preestablished B16 melanoma.     

Majority of Gliadin-Specific T-Cell Clones from Celiac Small Intestinal Mucosa Produce Interferon-γ and Interleukin-4

An abnormal mucosal cell-mediated immune response plays a fundamental role in the pathogenesis of celiac disease. To characterize locally infiltrating T cells, gliadin-specific T-cell clones were isolated from two treated celiac patients. Mucosal biopsies were cultured in vitro for 24 hr with a peptic-tryptic digest (PT) of gliadin. T-cell clones (TCC) were then isolated by limiting dilution. The production of interferon- (IFN-) and interleukin-4 (IL-4) was evaluated by ELISA in culture supernatants obtained after a short incubation with anti-CD3 and PMA, or with antigen. Twenty-two TCC were specific for gliadin and/or PT. All were CD3⁺, CD4⁺, CD8⁺, TCR ⁺. In one such clone the PT-specific response was inhibited by an anti-DQ, but not by an anti-DR antibody. Of the five gliadin-specific TCC examined, four produced IL-4 and high levels of IFN-; the remaining one initially produced only IL-4, but subsequently also IFN-. All clones obtained from the celiac mucosa, including the gliadin-specific ones, produced high levels of IFN-, in most cases with IL-4. This cytokine profile could explain most of the immunological features of the celiac mucosa.     

Increased frequency of CD4+PD-1+HLA-DR+ T cells is associated with disease progression in CLL

Chronic lymphocytic leukaemia (CLL) patients often have abnormal expansions of CD4⁺ and CD8⁺ T cells and this can be associated with progressive disease. To characterise the key T-cell populations involved in this phenomenon, we used flow cytometry and 11 phenotypic markers to study 74 CLL patients and 14 controls. T cells of CLL patients were more phenotypically complex than those of healthy controls with significant increases in the frequencies of CD4 and CD8 memory T cells expressing exhaustion-, activation- and senescence-associated markers. Multivariate analysis of 111 different T-cell subsets showed that high frequencies of four subsets (three CD8 and one CD4) were associated with shorter progression-free survival. The most significant association was with CD4⁺HLA-DR⁺PD-1⁺ T cells, and patients could be stratified into high- and low-risk groups based on the frequency of these T cells. The expansion of this CD4⁺ subset could not be accounted for by age, cytomegalovirus infection or increases in Treg cells. Overall, these results highlight two relatively simple biomarkers, percentage CD8⁺ and percentage CD4⁺PD-1⁺HLA-DR⁺ T cells, which can be used to risk-stratify CLL patients, independent of other tumour-associated markers. They also provide further evidence for the pivotal role of T cells in modulating the pathology of CLL.     

Effect of T-cell receptor VΒ-specific monoclonal antibodies on cyclophosphamide-induced diabetes mellitus in non-obese diabetic mice

Summary The expression of specific T-cell receptor gene segments by T lymphocytes appears to be critically important for the induction of several experimental autoimmune diseases mediated by these cells. We examined whether this situation also applied to non-obese diabetic mice by using various T-cell receptor V-specific monoclonal antibodies. No significant age- or sex-related differences were observed in V usage by peripheral and splenic T lymphocytes. CD8⁺ T lymphocytes among the islet-derived mononuclear cells isolated from 20-week-old female non-obese diabetic mice showed heterogeneity of their V gene usage. In order to examine the role of T lymphocyte subsets expressing specific T-cell receptor V gene segments in the development of diabetes mellitus, T-cell receptor V-specific monoclonal antibodies were administered to 10-week-old male non-obese diabetic mice treated with cyclophosphamide. None of the antibodies used could significantly diminish the incidence of cyclophosphamide-induced diabetes and the severity of insulitis [anti-V3 (11 of 22 mice became diabetic, 50%), anti-V5 (9 of 14, 64%), anti-V8 (9 of 21, 43%), anti-V11 (12 of 23, 52%), anti-V14 (7 of 12, 58%), and anti-V5 + anti-V11 (6 of 12, 50%)] when compared with control mice (12 of 21, 57%). In addition, there were no significant differences in T-cell receptor V usage between diabetic and non-diabetic cyclophosphamide-treated mice. These results suggest that five T-lymphocyte subsets expressing different T-cell receptor V gene segments, considered to be candidates involved in the pathogenesis of autoimmune diabetes, do not individually contribute to the development of cyclophosphamide-induced diabetes in non-obese diabetic mice.     

Recovery of antitumor CD4+ T cell responsiveness,suppressed in the tumor-bearing state,by release from tumor burden

The present study investigates the recovery of antitumor CD4⁺ T cell responsiveness, suppressed in the tumor-bearing state, following release of tumor burden. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1–3 weeks after the inoculation with CSA1M cells produced interleukin-2 (IL-2) and IL-4 upon in vitro cultures without addition of exogeneous tumor antigens. This lymphokine production was achieved through collaboration between anti-CSA1M CD4⁺ T cells and antigen-presenting cells (APC) that has been pulsed with CSA1M tumor antigens in vivo in the tumor-bearing state. The lymphokine-producing capacity gradually decreased as the tumor-bearing period increased, and spleen cells from mice at late (8–10 week) tumor-bearing stages produced reduced levels of lymphokines. Because APC in these cells exhibited enhanced capacities to present tumor antigens, the reduced responsiveness was ascribed to the dysfunction of CD4⁺ T cells themselves. However, removal of a tumor after 8 weeks resulted in a remarkable recovery of the lymphokine-producing capacities of whole spleen cells. In contrast to the reduction in CSA1M-antigen-presenting activity of APC following tumor resection, CD4⁺ T cells exhibited a reciprocal increase in their responsiveness to CSA1M antigens. The recovery of antitumor responsiveness was also observed in the in vitro cultures free from tumor burden; when spleen cells from mice at late tumor-bearing stages were pre-incubated for 1–2 days and re-cultured in fresh medium. They produced potent amounts of IL-2 and IL-4. These results indicate that the immunodysfunction of antitumor CD4⁺ T cells induced in the tumor-bearing state is not irreversible, and release from tumor burden results in almost complete recovery of the potent antitumor responsiveness they previously expressed.Abbreviations APC antigen-presenting cells - Th helper T cells - FITC fluorescein isothiocyanate - TGF transforming growth factor - IL interleukin This work was supported by Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture, Japan