A study on graft-versus-host reaction (GVHR) by Simonsen's splenomegaly assay. Cells and antigen systems involved in induction of GVHR

Cells and histocompatibility antigen systems involved in graft-versus-host reactions (GVHR) were analyzed using Simonsen's splenomegaly assay employing various combinations of donor and F1 hybrid recipients mice. Most of the cells proliferating in spleens of mice undergoing GVHR were J11d+, and had histological features of cells of the hematopoietic lineage. The proportions of CD3+ T cells were decreased in the spleens. Disparity at minor histocompatibility determinants of AKR, I-E and H-2D regions between B10.A(4R) donors and (4R X AKR) F1 recipients evoked only negligible GVHR. On the contrary, disparity at H-2K and/or I-A regions appeared to be sufficient to permit induction of full GVHR. When surface markers of donor spleen cells were analyzed, it was shown that Thy-1+ and/or MEL-14+ cells caused a strong effect on GVHR. Further, either CD4+ or CD8+ T cell subset could induce significant GVHR. However, synergistic influences of these two T cell subsets on one another in GVHR were observed. The present results raise the possibility of using Simonsen's assay along with a number of reagents to identify the contribution of subsets of T lymphocytes and in analyzing precise contributions of cellular components from both donor and recipient, and also of the target antigen systems of the recipient that contribute to early events involved in GVHR.     

Donor and recipient specific tolerance in cells from semi-allogeneic, H-2 subregion compatible or fully allogeneic bone marrow chimeras attributable to clonal deletion

Specificities of tolerance induced in allogeneic bone marrow (BM) chimeras which had been established by injecting allogeneic BM cells pretreated with anti-Thy-1 mAb alone (without complement (C)) were analyzed using Simonsen's splenomegaly assay. Lymphocytes from fully allogeneic, semi-allogeneic and H-2 subregion compatible BM chimeras were specifically unresponsive to donor and recipient antigens (Ag). However, cells from H-2 subregion compatible chimeras initiated as vigorously a GVHR in F1 recipient mice, which were disparate at H-2K and I-A regions, as did spleen cells of donor mice, which were incompatible at the entire H-2 and minor histocompatibility regions of the recipients. The donor cells from such chimeras that initiated these considerable GVHR were either CD4+ or CD8+ T cells. Furthermore, synergistic effects by the CD4+ and CD8+ T lymphocytes were also observed. We found no evidence for a suppressive mechanism(s) in maintenance of the specific tolerance in allogeneic chimeras. Further, when lymphoid cells from these chimeras were adoptively transferred to irradiated mice of the donor strain and maintained for 5 days in the absence of recipient Ag (tolerogen), the adoptively transferred cells were shown to retain their unresponsiveness to the recipient Ag. These results reveal that T lymphocytes from allogeneic BM chimeras prepared by our method had been specifically induced to a tolerant state to both donor and recipient Ag and that the major mechanism of induction and maintenance of long-lasting tolerance is attributable to clonal deletion of both CD4+ and CD8+ T cell subsets rather than to the development of a population of suppressor cells of any sort.     

Acute Graft-versus-Host Reaction in SCID Mice Leads to an Abnormal Expansion of CD8+ Vβ14+ and a Broad Inactivation of Donor T Cells Followed by a Host-Restricted Tolerance and a Normalization of the TCR Vβ Repertoire in the Chronic Phase

The persistence and selection of allogeneic CBA/J T lymphocytes were studied during graft-versus-host (GvH) reaction in immunodeficient C. B-17 SCID (SCID) mice. After neonatal injection the donor cells primarily migrated to the spleen plus lymph nodes (SL) and the thymus of the recipients. Thirteen days post engraftment, CD8⁺ cells in SL had increased five times in cell number with an 18-fold increase of CD8⁺ Vβ14⁺ cells, paralleled by clinical signs of GvH disease (GvHD). Donor lymphocytes from these mice were proliferative unresponsive to allogeneic Balb/c or C57B1/6 SL cells, whereas 8 weeks post injection the tolerance was confined to H-2^d specific donor cells. Here, spleens had a total cell content similar to untreated SCID mice but the average percentage of donor cells had reached 25%. Moreover, the CD4/CD8 cell ratio in the donor population in SL and thymus had changed to normal and the TCR Vβ repertoire was similar to that of the originally injected cells. Following secondary transfer into syngeneic CBA/Ca nu/nu recipients donor cells regained a significant but reduced response to H-2^d stimulators indicating that the antigen specific tolerance of allogeneic donor cells in the SCID mice was due, at least in part, to a reversible state of anergy.     

Intrathymic maturation of CD4+ T-lymphocytes in an MHC class II deficient transplant model

Major histocompatibility complex (MHC) class II knockout (class II^-) mice fail to generate CD4⁺ CD8^- T-lymphocytes. We were interested in determining whether these class II^- mice could be reconstituted with CD4⁺ CD8^- T-lymphocytes following marrow transplantation from normal (class II⁺) donors. Transplantation of class II⁺ marrow into lethally irradiated class II^- recipients failed to generate peripheral CD4⁺ CD8^- T-lymphocytes. Unexpectedly, however, transplantation of class II^- marrow into class II⁺ recipients also resulted in a deficiency of CD4⁺ CD8^- cells. Analysis of intrathymic T cells showed normal distribution of CD4 and CD8 single and double positive or negative thymocytes in normal recipients, while class II^- recipients always lacked CD4⁺ CD8^- T cells intrathymically. These results suggest, therefore, that T-cell maturation in mice requires the presence of MHC class II antigens not only in the thymus but also on immature, marrow-derived pre-thymocytes.     

Lymphocytic Choriomeningitis Virus Infection is Associated with Long-Standing Perturbation of LFA-1 Expression on CD8+ T Cells

Flow cytometric analysis of splenocytes from mice infected with lymphocytic Choriomeningitis virus revealed marked and long-standing up-regulation of LFA-1 expression on CD8⁺, but not on CD4⁺ T cells. Appearance of CD8⁺ T cells with a changed expression of adhesion molecules reflected polyclonal activation and expansion which was demonstrated not to depend on CD4⁺ T cells or their products. Cell sorting experiments defined virus-specific CTL to be included in this population (LFA-1^hiMEL-14^lo), but since about 80% of splenic CD8⁺ T cells have a changed phenotype, extensive bystander activation must take place; this is indicated also by the finding that CD8⁺LFA-l^hi cells transiently express several markers of cellular activation, e. g. transferrin receptor, IL-2Rα and β. Analysis of cells from the cerebrospinal fluid of mice infected intracerebrally showed that virtually all T cells present belonged to the CD8LFA-lhi subset and, correspondingly, the ligand ICAM-1 was found to be up-regulated on endothelial cells in the inflamed meninges. Preincubation of LCMV-primed donor splenocytes with anti-LFA-1 markedly inhibited the transfer of virus-specific delayed-type hypersensitivity to naive recipients. Together, these findings indicate that up-regulation of LFA-1 expression is a critical factor involved in directing activated CD8⁺ T cells to sites of viral infection.     

Enhanced T Lymphocyte Expression of LFA-1, ICAM-1, and the TNF Receptor Family Member OX40 in HgCl2-Induced Systemic Autoimmunity

Injection of mercuric chloride into Brown Norway (BN) rats induces a T lymphocyte-dependent autoimmune syndrome. In order to investigate whether modification of adhesion and costimulatory molecules on T lymphocytes may be involved in early T lymphocyte activation by HgCl₂, the authors analysed expression of these molecules in peripheral lymph node cells from BN rats at day 4 after injection of HgCl₂. Tri-colour flow cytometry was performed for expression analysis within CD45RC-defined subsets of CD4⁺ and CD8⁺ cells. Compared to control rats, HgCl₂-exposed rats showed increased numbers of lymphocytes, especially of T lymphocyte blast cells. The levels of LFA-1 expression as well as the fractions of ICAM-1⁺ cells were significantly increased in all CD45RC-defined subsets of CD4⁺ and CD8⁺ cells. Within the CD4⁺CD45RC^lo T lymphocyte population, HgCl₂-injected rats showed a highly significant increase in the number of cells expressing OX40, which is a member of the TNF receptor family. Moreover, only CD4⁺CD45RC^lo blast cells of HgCl₂-exposed rats showed decreased expression of CD43, increased expression of CD49d and decreased numbers of CD26⁺ cells. The results indicate that induction of autoimmunity by HgCl₂ in BN rats is associated with altered expression of T lymphocyte costimulatory molecules, predominantly on CD4⁺CD45RC^lo cells, which may be caused by a direct effect of HgCl₂ on these cells, and may precipitate further activation of T and B lymphocytes by HgCl₂     

Abrogation of negative selection by GVHR induced by minor histocompatibility antigens or H-2D antigen alone

Allogeneic bone marrow chimeras were prepared by donor and recipient combinations that differed in minor histocompatibility loci or H-2D locus alone. When 1 x 10(5) splenic T cells were inoculated in addition to T cell-depleted bone marrow cells (1 x 10(7)), clinically detectable GVHR was induced. In these GVHR chimeras, substantial numbers of T cells reactive to either donor or recipient antigens were both phenotypically and functionally detected. The mechanisms underlying the abrogation of intrathymic negative selection are discussed.     

Unresponsiveness of CD4+ T Cells from a Non-Responder Strain to HgCl2 is Not Due to CD8+-Mediated Immunosuppression: an Analysis of the Very Early Activation Antigen CD69

Injections of HgCl₂ lead to autoimmune manifestations in genetically predisposed rats and mice. In this study, the authors examined the responsiveness of T subsets from different mouse strains to HgCl₂ by tracing their expression of the very early activation antigen CD69. The authors found increased expression of the CD69 antigen on CD4⁺ T cells from the responder A.SW and BALB/c mice, but not on CD4⁺ T cells from the non-responder DBA/2 mice, indicating an activation of T helper cells in the responder strains. However, the CD69 antigen was induced on CD8⁺ T cells from all strains irrespective whether they were susceptible or resistant to mercury-induced autoimmunity. Since CD8⁺ T cells have been described as mediating immunosuppression and as being responsible for the resistance to autoimmune induction by mercury, the authors tested whether CD8⁺ T cells inhibited the activation of CD4⁺ T cells by HgCl₂ in the non-responder strains. However, there was no evidence for a suppressive role of CD8⁺ T cells from the DBA/2 mice in the response to HgCl₂. The findings indicate that T helper cells play a central role in the immunological effects of HgCl₂ and unresponsiveness of T helper cells in the nonresponder strains is not due to CD8⁺-mediated immunosuppression.     

Strict Adherence to a Common Rank Order of T-Cell Receptor Vβ Usage in Human Leucocyte Antigen Disparate Individuals

In order to investigate the T-cell receptor (TCR) Vβ usage in different T-cell subsets, the authors performed flow cytometric analyses using a large panel of TCR Vβ-specific monoclonal antibodies on CD4⁺, CD8⁺ CD28⁺ and CD8⁺ CD28⁻ T cells from 15 random blood donors, six umbilical cords and seven human leucocyte antigen (HLA) identical non-twin sibling pairs. The authors found that the proportion of T cells expressing each Vβ gene product was similar within CD4⁺ and CD8⁺ CD28⁺ T cells from all samples studied. For these T-cell subsets a rank order of Vβ usage could be identified which was adhered to by all donors. In contrast, within CD8⁺ CD28⁻ T cells a wide variation of Vβ usage was found between individuals, and no rank order correlation could be detected. Members of HLA identical sibling pairs were found to be no more similar in their usage of Vβ gene products than pairs of HLA disparate random blood donors. Groups of individuals sharing HLA antigens were no different from the groups not sharing such antigens in their usage of Vβ segments. The results suggest that HLA polymorphisms play no more than a minor part in determining TCR Vβ usage.     

The CD4+CD8+ and CD4+ Subsets of FOXP3+ Thymocytes Differ in their Response to Growth Factor Deprivation or Stimulation

The timing of thymic regulatory T (Treg) cell commitment remains unclear. Specifically, there is disagreement as to whether the CD4⁺CD8⁺ FOXP3⁺ thymocytes are precursors of mature CD4⁺ FOXP3⁺ Treg cells, or an independent Treg cell lineage. We reasoned that precursors should be more susceptible to apoptosis than mature Treg cells, and tested this by growth factor removal and anti-CD3 stimulation. Both treatments resulted in an increase of CD4⁺ FOXP3⁺ thymocytes, whereas the frequency of CD4⁺CD8⁺ FOXP3⁺ thymocytes decreased significantly. These changes were accompanied by an increase of annexin⁺ apoptotic cells. Both of these FOXP3⁺ subsets expressed higher levels of Bcl-2 and BIM than other thymocytes, and while in our setting expression of BIM seemed to predispose the cells to apoptosis, Bcl-2 had no apparent protective effect. These results indicate that CD4⁺CD8⁺ FOXP3⁺ thymocytes are more susceptible to apoptosis than mature CD4⁺ FOXP3⁺ Treg cells. This is consistent with the view that they are still immature and thus likely to represent a precursor population.     

T-Lymphocyte Functions in Acute Leukaemia Patients with Severe Chemotherapy-Induced Cytopenia: Characterization of Clonogenic T-Cell Proliferation

Intensive chemotherapy for acute leukaemia is followed by a period of severe chemotherapy-induced leukopenia. We used a limiting dilution assay to investigate whether remaining CD4⁺ and CD8⁺ T lymphocytes derived from such leukopenic patients could be activated and undergo clonogenic proliferation. The activation signal in our model was accessory cells (irradiated normal peripheral blood mononuclear cells) + phytohaemagglutinin (PHA) + interleukin-2 (IL-2). During severe leukopenia a majority of circulating lymphocytes were CD4⁺ T cells. Clonogenic proliferating T lymphocytes were detected for all patients. Higher frequencies of clonogenic cells were detected in the CD8⁺ subset as compared to the CD4⁺ subset. However, for both subsets frequencies of proliferating cells were decreased compared with healthy individuals. The CD4⁺ and CD8⁺ lymphocytes were also capable of proliferation in response to alloactivation, and accessory cells mainly containing acute myelogenous leukaemia blast were efficient as accessory cells for activation. For the CD4⁺ cells, increased proliferation was detected in the presence of acute myelogenous leukaemia (AML) blasts compared with normal accessory cells. Based on our results we conclude that: (1) although acute leukaemia patients with therapy-induced leukopenia have both a quantitative and a qualitative T-cell defect, (2) the remaining T-cell population includes a subset capable of clonogenic proliferation. However, (3) proliferation of the clonogenic CD4⁺ cells can be modulated by AML blasts.     

Use of Immobilized HLA-A2:Ig Dimeric Proteins to Determine the Level of Epitope-Specific, HLA-Restricted CD8+ T-Cell Response

A novel assay to assess antigen-specific cytokine release from stimulated CD8⁺ T cells derived from the mucosal and peripheral blood compartments has been developed and standardized using the influenza A virus matrix protein (MP) peptide, GILGFVFTL. This technology is based on the capacity for the human leucocyte antigen (HLA)-A2:Ig dimeric protein to stimulate CD8⁺ T cells in a major histocompatibility complex (MHC) class I-restricted fashion without the necessity for antigen presenting cells (APC). This assay has been optimized utilizing a 9-amino acid residue (9mer) peptide, the optimal peptide length for presenting an epitope to CD8⁺ T cells. Compared to existing assays, this more sensitive and specific methodology requires fewer cells, enabling easier and more accurate monitoring of the CD8⁺ T-cell response in biological compartments, such as the mucosa during the course of viral infection and may be utilized to assess epitope-specific CD8⁺ T-cell responses in vaccine trials.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

Proliferation of C3HXCBA Hybrid Lymphocytes in the Spleens of Irradiated CBA Mice: Linkage to the Mls-Gene

Lymphocytes from C3HXCBA F₁ mice, lacking delectable Mls-antigens, can specifically inactivate T-cell clones from the H-2-idcmical strain CBA, which are reactive against the C3H-Determined Mls-antigen. Such F₁ lymphocytes also proliferate in the spleens of irradiated CBA mice. The age dependence of the mixed lymphocyte culture stimulatory capacity and the capacity to proliferate in irradiated CBA recipients of lymphocytes from C3HXCBA F₁ mice were compared. It was observed that appearance of Mls-bearing cells is preceded by the appearance of F₁ cells with strong proliferate capacity in irradiated CBA recipients. The in vivo proliferative response is also dependent on the age of the recipient. The varying response is not reflected in the quantity ofanli-C3H Mls-reactive cells in CBA mice. A strong linkage between the genes controlling the above proliferative and stimulatory capacities of C3HXCBA lymphocytes was found.     

CD4+CD25+ regulatory cells in acquired MHC tolerance

Summary: Tolerance to self-antigens is an ongoing process that begins centrally during T-cell maturation in the thymus and continues throughout the cell's life in the periphery by a network of regulated restraints. Remaining self-reactive T-cells that escape intrathymic deletion may be silenced within the peripheral immune system by specialized regulatory CD4⁺ cells. By analogy, regulatory CD4⁺ cells that control immunity to "acquired self" should arise in circumstances where the immune system acquires tolerance to foreign MHC, such as the tolerance that develops following the exposure to foreign MHC antigens during the neonatal period. We have used this classic model of neonatal tolerance to examine the role of regulatory CD4⁺ cells in acquired tolerance to disparate class I and class II MHC. Adoptive transfer of unfractionated but not CD4⁺-depleted spleen cells from neonatal tolerant mice into SCID recipients inhibited skin graft rejection by immunocompetent CD8⁺ T cells. Using 5-bromo-2'-deoxyuridine incorporation, standard cytotoxic T-lymphocyte assays, short-term interferon-γ ELISPOT, and intracellular FACS analysis to study CD8⁺ T-cell effector function, we demonstrated that neonatal tolerant mice contain CD4⁺CD25⁺ cells that suppress the development of anti-donor CD8⁺ T-cell responses in vitro . We conclude that regulatory CD4⁺CD25⁺ cells initiate and/or maintain tolerance by preventing the development of CD8⁺ T-cell alloreactivity.     

Participation of the CD69 Antigen in the T-Cell Activation Process of Patients with Systemic Lupus Erythematosus

Accumulating evidence has implicated T cells in the pathogenesis of systemic lupus erythematosus (SLE). The CD69 antigen is an integral membrane protein rapidly induced on the surface of activated lymphocytes. We obtained CD4⁺ and CD8⁺ T cells from normal subjects and patients with SLE. The percentage of CD69 expression in freshly isolated cells and after in-vitro incubation with mitogens was quantified by three-colour immunofluorescent staining. Expression of this protein was increased in both CD4⁺ and CD8⁺ T-cell subsets from SLE patients when compared with normal cells, although the difference was significant only in the CD8⁺ T-cell subset ( P  = 0.05). Cellular activation increased CD69 expression. When stimulated with anti-CD2/CD2R or phytohaemagglutinin (PHA), the percentage and absolute numbers of CD69⁺ cells were lower in patients than in controls. Addition of anti-interleukin (IL)-10 monoclonal antibody (MoAb) increased the percentage of in-vitro CD69 expression in SLE cells. These results suggest that the peripheral blood lymphocytes from patients with SLE have an intrinsic defect that alters their activation process, including the expression of CD69, and might explain some of the T immunoregulatory abnormalities observed in these patients.     

T-Cell Regulation of CD5+ B-Cell Activity in Normal Mice

The differentiation of plaque-forming cell (PFC) precursors against bromelain-treated syngeneic erythrocytes (Br MRBC) into PFC induced in vitro by LPS is down-regulated by nylon non-adherent (nylon-passed–NP) T cells and by nylon adherent (NA) T cells, NA T cells are more potent inhibitors than NP T cells. This regulatory activity of NA and NP T cells results from an interaction between CD4⁺ radioresistant and CD4⁺ radiosensitive T cells. Furthermore CD4⁺ T cells from the NA fraction but not from the NP fraction are activated cells: their inhibitory activity is abrogated after preincubation with cycloheximide. These results are discussed within the overall framework of T-cell regulation of autoimmune anti-Br MRBC B-cell subsets.     

The impact of T-cell immunity on ovarian cancer outcomes

Summary: Ovarian cancer remains a challenging disease for which improved treatments are urgently needed. Most patients present with advanced disease that is highly responsive to surgery combined with platinum- and taxane-based chemotherapy, with a state of minimal residual disease being achieved in many cases. However, chemotherapy-resistant recurrent tumors typically appear within 1–5 years and are ultimately fatal. Recently, several groups have shown that ovarian tumors are often infiltrated by activated T cells at the time of diagnosis, and patients with dense infiltrates of CD3⁺CD8⁺ T cells experience unexpectedly favorable progression-free and overall survival. Other cell types in the immune infiltrate oppose anti-tumor immunity, including CD4⁺CD25⁺FoxP3⁺ regulatory T cells, CD8⁺ regulatory T cells, macrophages, and dendritic cells. The composition of immune infiltrates is shaped by the expression of cytokines, chemokines, antigens, major histocompatibility complex molecules, and costimulatory molecules. The relationship between these various immunological factors is reviewed here with a strong emphasis on outcomes data so as to create a knowledge base that is well grounded in clinical reality. With improved understanding of the functional properties of natural CD8⁺ T-cell responses to ovarian cancer, there is great potential to improve clinical outcomes by amplifying host immunity.     

Soluble CD23 (sCD23) serum levels and lymphocyte subpopulations in peripheral blood in rhinitis and extrinsic and intrinsic asthma

To determine serum levels of IgE and sCD23 and lymphocyte subpopulations, we studied 37 control subjects and 84 patients (27 with allergic rhinitis, 27 with extrinsic asthma, and 30 with intrinsic asthma). A rise in surface CD23 on B and monocyte cells and sCD23 serum levels was exhibited by patients with rhinitis and extrinsic asthma. Unexpectedly, in intrinsic asthmatic patients, high CD23 expression on monocytes and high sCD23 levels were seen that did not result in IgE production. It appears that CD23, in its soluble form, could be a good disease marker, especially in asthma. Atopic patients yielded a significantly lower proportion of CD4⁺ T cells than intrinsic asthmatic patients and normal persons. Otherwise, CD4⁺ CD29⁺ CD45RA - and CD4⁺ CD29 – CD45RA ⁺ T-cell subsets were significantly decreased in all patient groups.