E.A.J.M. Goulmy

A Viral ER-Resident Glycoprotein Inactivates the MHC-Encoded Peptide Transporter

Human cytomegalovirus inhibits peptide import into the endoplasmic reticulum (ER) by the MHC-encoded TAP peptide transporter. We identified the open reading frame US6 to mediate this effect. Expression of the 21 kDa US6 glycoprotein in human cytomegalovirus-infected cells correlates with the inhibition of peptide transport during infection. The subcellular localization of US6 is ER restricted and is identical with TAP. US6 protein is found in complexes with TAP1/2, MHC class I heavy chain, beta2-microglobulin, calnexin, calreticulin, and tapasin. TAP inhibition, however, is independent of the presence of class I heavy chain and tapasin. The results establish a new mechanism for viral immune escape and a novel role for ER-resident proteins to regulate TAP via its luminal face.

Immunogenetics of human minor histocompatibility antigens : their polymorphism and immunodominance

Minor Histocompatibility (mH) antigens are polymorphic endogenously synthesized products that can be recognized by alloreactive T cells in the context of major histocompatibility complex molecules. In transplant situations where tissue donor and recipient are matched for HLA, mH antigens may trigger strong cellular immune responses. To gain insight into the polymorphism of mH antigens we studied their frequencies in the healthy population. Five HLA class I restricted mH antigens recognized by distinct cytotoxic T-cell (CTL) clones were used in the population genetic analysis consisting of a panel (N=100) of HLA typed target cells. Three mH antigens showed phenotype frequencies of 69% or higher, this contrasted the frequencies of two other mH antigens with 16 and 7% respectively. To gain insight into the “functional” polymorphism of the T-cell response to mH antigens, we analyzed the specificity of CTL clones within individuals. Three out of five individuals investigated shared a CTL response to one single HLA-A2 restricted mH antigen. These results indicate limited allelic polymorphism for some mH antigens in the healthy population and are suggestive of the existence of immunodominant human mH antigens.

Rejection of bone-marrow graft by recipient-derived cytotoxic T lymphocytes against minor histocompatibility antigens.

A female patient showed rejection of a T-lymphocyte-depleted bone-marrow graft from her phenotypically HLA-identical father. Before bone-marrow transplantation, there was strong recipient anti-donor cellular cytotoxic reactivity directed against several minor histocompatibility (mH) antigens, including the male-specific H-Y antigen. After conditioning treatment, no recipient anti-donor cytotoxic activity could be detected, and good graft function was shown a month after transplantation. Thereafter, however, graft function deteriorated rapidly, while recipient-derived anti-donor cellular cytotoxic reactivity, against similar mH antigens, reappeared. The recipient-derived cytotoxic T lymphocytes could completely inhibit growth of donor haemopoietic progenitor cells both before and after bone-marrow transplantation. Thus, cytotoxic T lymphocytes can survive very intensive conditioning regimens, and residual recipient cytotoxic T lymphocytes directed against mH antigens expressed on donor haemopoietic progenitor cells may cause graft rejection after HLA-identical T-lymphocyte-depleted bone-marrow transplantation.

Serum cytokine levels after HLA-identical bone marrow transplantation.

BACKGROUND Altered profiles of cytokine production are observed after bone marrow transplantation (BMT). The presence of certain cytokines in serum can be indicative for BMT-related complications, such as graft-versus-host disease (GVHD) and infections. The putative correlation between abnormal serum cytokine levels and BMT-related complications was further analyzed in this retrospective study. METHODS Serum levels of a panel of cytokines and cytokine-associated molecules (i.e., interleukin [IL]-1alpha, IL-1beta, IL-4, IL-6, IL-10, IL-12, IL-1 receptor antagonist, and the soluble a chain of the IL-2 receptor [sIL-2R alpha]) were assessed in 329 sera of 46 patients who had undergone HLA-identical or autologous BMT. Serum cytokine levels of the BMT donor and of the patients before BMT and at different time points during the post-BMT period were measured. The results were correlated with relapse, acute GVHD, chronic GVHD, and infections. RESULTS Serum levels of IL-1alpha, IL-1beta, IL-4, and IL-12 were undetectable. The transplantation regimen itself causes a significant rise in IL-10 and sIL-2R alpha levels in patients receiving allogeneic bone marrow. In the post-BMT period, increased IL-6 serum levels were significantly correlated with infections. Increased IL-10 levels were significantly correlated with acute graft-versus-host disease, chronic GVHD, and infections. Increased sIL-2R alpha levels were correlated with chronic GVHD, as were IL-1 receptor antagonist levels. CONCLUSIONS During the post-HLA-identical BMT period, the serum cytokine levels of IL-6 were enhanced during infections, whereas the sIL-2R alpha levels were increased during chronic GVHD. The serum levels of IL-10 and of the cytokine-related molecule IL-1ra were enhanced during both infections and chronic GVHD. These results further substantiate the complex cytokine cascade that is initiated by the conditioning regimen and that evolves further in reaction to BMT-related complications and their treatments.

The effects of stress and relaxation on the in vitro immune response in man; a meta-analytic study.

The purpose of the present meta-analytic study was to combine and integrate the results of stress and relaxation studies for their reported changes in the in vitroimmune response. Twenty-four stress studies and 10 relaxation studies with a (quasi)-experimental design with pre- and postintervention measurements were selected. Twenty immunological variables tested in stress studies and five immunological variables tested in relaxation studies could be further analyzed. The meta-analysis of the results of the stress studies indicated that the observed changes in interleukin-2 receptor expression on lymphocytes and antibody titers against Epstein Barr virus (EBV) were consistent for the direction of change and globally significant, whereas the observed changes in percentage of natural killer (NK) cells, salivary immunoglobulin A (sIgA) concentration, and antibody titers against Herpes simplexvirus (HSV) were not consistent and not significant. Analysis of the results of the relaxation studies indicated that the observed changes in sIgA concentration were consistent for direction of change and significant, the results for white blood cell count were consistent but not significant, and the results for percentage of monocytes were neither consistent nor significant.

Month-related variability in immunological test results; implications for immunological follow-up studies.

This longitudinal study was originally designed to detect changes in the in vitro immune response of healthy subjects as a result of a psychological intervention. In this study a significant proportion, about 70%, of the immunological variability in the test results was accounted for by the differences in immunological response levels of the subjects. Apart from this between‐subject‐effect, a significant proportion of the variability in test results was related to the month of data sampling. The month‐effect was computed in such a way that the between‐subject variation was taken into account. This resulted in a more accurate estimation of the month‐effect. Even after correction for the intervention, i.e. the defence of the PhD thesis, the effect of month of data sampling remains significant for mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, percentage of CD4 and CD8 cells, and for the response to the mitogens phytohaemagglutinin, pokeweed mitogen and concanavalin A as well as the results for the mixed lymphocyte culture for one pool out of three. In contrast, no significant month‐effect was observed for the whole blood cell counts, for the differential white blood cell counts as determined by monoclonal antibody staining for cell surface markers CD3, GD16, TAG and OKM1, nor for the immunoglobulin IgM and IgG serum levels. Likewise the cell‐mediated lympholysis activities measured against three pools of stimulator cells remained unaltered. We discuss the implications for future immunological follow‐up studies of the observation that a significant proportion of the variability in immunological test results is related to differences between subjects and to the month of data sampling.

Differential recognition of the serologically defined HLA-A2 antigen by allogeneic cytotoxic T Cells

Human alloimmune cytotoxic T cells, sensitized selectively against the HLA-A2 antigen, were tested on a panel of selected target cells. Five HLA-A2 positive outlier cells could be identified. These outlier cells were only weakly lysed by HLA-A2 specific CTLs, although they were serologically indistinguishable from the other HLA-A2 positive, strongly lysed target cells. Furthermore, it was found that the outlier cells were poor cold target inhibitors in contrast to the other HLA-A2 positive target cells, which showed adequate inhibition of specific lysis of HLA-A2 positive target cells. Population studies indicate that the frequency of such HLA-A2 outlier cells may be approximately 10%.

Deletion mapping of H-Y antigen to the long arm of the human Y chromosome

A gene encoding or controlling the expression of the H-Y transplantation antigen was previously mapped to the human Y chromosome. We now report the sublocalization of this gene on the long arm of the human Y chromosome. Eight patients with Y-chromosomal abnormalities were examined with a series of existing and new DNA markers for the Y chromosome. The resulting deletion map was correlated with H-Y antigen expression. We conclude that the H-Y antigen gene maps to a portion of deletion interval 6 that is identified by specific DNA markers.

Human minor histocompatibility antigens.

For over five decades minor histocompatibility (mH) antigens have contmued to fascmate immimologists, mainly because of their minor but distinct role in transplantation but no less because of their as yet unknown nature In the 1970s lt became evident that mH antigens were recognized in a MHC restncted fashion, an important feature which was poorly understood at that time Through the innovative work on the MHC class I crystals, significant insight into the nature of mH antigens as MHC-bound peptides has recently been obtained Thanks to extensive munne and human studies, more knowledgc, though still the tip of the lceberg, has been gathered —

Escherichia coli -nitroreductase suicide gene control of human telomerase reverse transcriptase-transduced minor histocompatibility antigen-specific cytotoxic T cells

Summary:Adoptive immunotherapy with ex vivo generated cytotoxic T lymphocytes (CTLs) is applied for the treatment of leukemia relapses or viral infections after allogeneic stem cell transplantation. A common problem of adoptive immunotherapy strategies is the ex vivo expansion of the generated T cells to sufficient numbers. CTLs can be efficiently expanded by ectopic expression of the human telomerase gene (hTert). However, hTert transduction may also increase the chance for malignant transformation. Therefore, we explored the feasibility of suicide gene control of ex vivo generated CTLs expanded through the ectopic expression of hTert. To this end, we compared the efficacy of the new Escherichia coli-nitroreductase (E. coli-Ntr) suicide gene with the well-known herpes simplex virus-thymidine kinase (HSV-Tk). Introduction of hTert provided the transduced CTLs with a distinct growth advantage over the nontransduced CTLs. The hTert-E. coli-Ntr double-transduced CTLs retained their antigen-specific functions. Treatment of hTert-E. coli-Ntr double-transduced CTLs with metronidazole significantly inhibited the proliferation to a similar extent to the treatment of hTert-HSV-Tk double-transduced CTLs with ganciclovir. This is the first application of the E. coli-nitroreductase gene for the elimination of human T cells with metronidazole.