Production of IL-10 by alloreactive sibling donor cells and its influence on the development of acute GVHD.

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation. Pretransplant conditioning regimes cause release of proinflammatory cytokines that stimulate alloreactive donor T cells to attack recipient tissues. IL-10 has been shown to directly downregulate CD4+ T cells by suppressing IL-2 secretion and a critical role played by regulatory T cells has been demonstrated in animal models. One defining cytokine profile for regulatory T cells is the production of IL-10. Release of specific cytokines (IL-10, IL-4 and IFN-gamma) was detected using ELISPOT technology, following stimulation of donor peripheral blood mononuclear cells by recipient (human leukocyte antigen-matched sibling) alloantigen or by mitogen. Correlation between the frequency of cytokine-releasing cells and the development of acute GVHD was investigated. A high frequency of donor cells producing IL-10 in response to recipient alloantigen stimulation correlated with absence of acute GVHD after bone marrow transplant (BMT), while low frequency was strongly associated with severe GVHD. This study presents strong evidence that estimating the frequency of donor alloreactive cells producing IL-10 in response to recipient antigens will provide valuable information prior to BMT regarding potential transplant outcome.

Influence of FcgammaRIIA and MBL polymorphisms on severe acute respiratory syndrome.

Polymorphisms of human Fc gamma-receptor IIA (FcgammaRIIA) and mannose-binding lectin (MBL) genes have been associated with susceptibility to or severity of some infectious diseases. In order to investigate whether these genetic factors might influence susceptibility to infection with the severe acute respiratory syndrome-associated coronavirus (SARS-Cov) as well as the course and severity of the infection, we evaluated polymorphisms of FcgammaRIIA and MBL genes in DNA samples from a group of approximately 180 people from Hong Kong who were infected with SARS-Cov. These included 132 patients who had moderate course of SARS infection (home subgroup), 26 patients with a severe course requiring treatment in an intensive care ward (ICU subgroup) and a subgroup of 22 patients who died from SARS (deceased subgroup). A total of 200 normal blood donors from the same region were used as controls. A significant association was found between the FcgammaRIIA-R/R131 genotype and a severe course of SARS, with higher frequency of homozygosity for FcgammaRIIA-R/R131 in the ICU subgroup of SARS patients when compared with controls (P=0.03; odds ratio: 3.2; 95% confidence interval: 1.1-9.1). In comparison with controls, a significant difference in linear trend distribution of FcgammaRIIA genotypes was seen among the severe SARS patients (ICU and deceased subgroups) without co-morbidity, and the incidence of FcgammaRIIA-H/H131 was lower in these patients as well. There were no significant differences in MBL genotypes and allele frequencies among SARS patients and controls. The study reveals that in addition to age and co-morbidity, FcgammaRIIA polymorphism of individuals may also influence outcome after infection with the SARS-Cov.

Interleukin-2-producing helper T lymphocyte precursor frequency and rejection: a longitudinal cellular study after cardiac transplantation.

Interleukin-2-producing helper T lymphocyte precursors (HTLp) in the recipient recognize donor alloantigen expressed by the transplanted organ. The frequency of these reactive cells in the peripheral blood was determined and correlated with rejection episodes. Endomyocardial biopsy is generally used to quantify cardiac allograft rejection and guide immunotherapy. While non-invasive techniques have been investigated, none of these has demonstrated sufficient sensitivity or specificity to replace myocardial biopsy. Twelve cardiac transplant recipients were assessed over a 1 year period using limiting dilution analysis, to determine the frequency of HTLp in response to cadaver donor splenocytes. The IL-2-dependent mouse cell line CTLL-2 was used to measure the IL-2 present and the precursor frequency was calculated using maximum likelihood estimation. In the months immediately post transplantation, six of the 12 recipients displayed an association with increases in HTLp frequency, which preceded histologically detectable rejection. The second six recipients had fewer rejection episodes and achieved 'acceptance' of their graft sooner. Once 'acceptance' was achieved, the association between IL-2 HTLp frequency and rejection was no longer apparent. The ability to identify two groups of patients on the basis of IL-2 HTLp frequency clearly highlights the heterogenous nature of the response to the graft and this emphasizes the need to monitor other cytokines, which may influence the functioning of effector T cells.

A large, single center investigation of the immunogenetic factors affecting liver transplantation.

BACKGROUND: Reports on the relevance of immunogenetic factors in liver transplantation are often conflicting or inconclusive. We have, therefore, investigated a range of factors that may underlie liver graft survival. METHODS: The influences of HLA, flow cytometric, and enhanced cytotoxic crossmatching and immunoglobulin (Ig)A levels on graft survival, and acute and chronic rejection were investigated for a single center involving 446 patients over 13 years. RESULTS: The effect of HLA mismatching on graft survival was significant (P<10(-2)) and was reversed in recipients with autoimmune diseases (P<0.5x10(-2)), whereas the effect of HLA mismatches on the level of acute rejection was detrimental in all recipients. There was a significant effect of a positive cytotoxic crossmatch on 3-month (P<10(-5)) and 1-year (P<10(-4)) graft survival, and an additional effect of the flow cytometric crossmatch was seen for chronic rejection (P<10(-2)) and acute rejection (P<10(-2)). Recipients with HLA-A1,B8,DRB1*0301 had higher levels of acute rejection (P<0.5x10(-2)), and recipients who received an ABO compatible-nonidentical transplant have a significantly higher risk (P<10(-2)) of developing chronic rejection. Finally, the beneficial effect of high serum IgA and, specifically, IgA anti Fab, seen in renal transplants was not evident in liver transplants, and in fact the opposite may be true, at least for acute rejection (P<0.5x10(-2)). CONCLUSIONS: By separating the recipients with autoimmune disease from other patients and by including acute and chronic rejection as outcome parameters, we have used the power of a large single-centre study to delineate the significance of some of the important immunogenetic factors involved in liver transplantation.

HLA and other host factors in transfusion-acquired HIV-1 infection.

The host and viral factors that underlie infection with HIV-1 vary considerably with some individuals progressing to AIDS within 3 to 5 years after infection, whereas others remain clinically asymptomatic for over 10 years. Host factors that may contribute to disease progression include HLA and allelic variants of the chemokine receptors CCR5 and CCR2, which have been shown to influence both long-term survival and rapid progression. In this study, we have examined the contribution of HLA and polymorphisms in CCR5 and CCR2 to long-term survival in transfusion-acquired HIV-1-infected individuals. We have found a higher number of HLA-A32 and -A25 alleles but a lower number of the HLA-B8 allele in the study group compared with the frequencies seen in the HIV-1-negative Australian caucasian population. However, there was no apparent contribution by allelic variants of CCR5 and CCR2 to long-term survival and the combined influence of HLA and CCR polymorphisms could not be evaluated in this relatively small (n = 20) group of study subjects. The results of this work support a role for HLA in long-term nonprogression though the presence in the Sydney Blood bank Cohort of nef-defective HIV-1 may confound associations between certain HLA alleles and long-term survival in the face of infection with HIV-1.

Effect of long-term infection with nef-defective attenuated HIV type 1 on CD4+ and CD8+ T lymphocytes: increased CD45RO+CD4+ T lymphocytes and limited activation of CD8+ T lymphocytes.

Members of the Sydney Blood Bank Cohort (SBBC) have been infected with an attenuated strain of HIV-1 with a natural nef/LTR mutation and have maintained relatively stable CD4+ T lymphocyte counts for 14-18 years. Flow cytometric analysis was used to examine the phenotype of CD4+ and CD8+ T lymphocytes in these subjects, including the immunologically important naive (CD45RA+CD62L+), primed (CD45RO+), and activated (CD38+HLA-DR+ and CD28-) subsets. The median values were compared between the SBBC and control groups, comprising age-, sex-, and transfusion-matched HIV-1-uninfected subjects; transfusion-acquired HIV-1-positive LTNPs; and sexually acquired HIV-1-positive LTNPs. Members of the SBBC not only had normal levels of naive CD4+ and CD8+ T lymphocytes, but had primed CD45RO+ CD4+ T lymphocytes at or above normal levels. Furthermore, these primed cells expressed markers suggesting recent exposure to specific antigen. SBBC members exhibited variable activation of CD8+ T lymphocytes. In particular, SBBC members with undetectable plasma HIV-1 RNA had normal levels of activated CD8+ T lymphocytes. Therefore, the result of long-term infection with natural nef/LTR mutant HIV-1 in these subjects suggests a decreased cytopathic effect of attenuated HIV-1 on susceptible activated CD4+ T lymphocyte subsets in vivo, and minimal activation of CD8+ T lymphocytes.

The Sydney Blood Bank Cohort: a case-control study using a transfused HIV-1 seronegative group.

PURPOSE: To compare the immunological function of the Sydney Blood Bank Cohort (SBBC), a unique group of individuals who were all infected with a similar, attenuated strain of HIV-1, with a matched HIV-1 seronegative control group. To establish whether the asymptomatic state of the SBBC, in 1996, was likely to continue, and whether the SBBC were free from immunological signs of disease progression. METHODS: A prospective case-control design using a matched transfused HIV-1 seronegative control group. Immunological testing was performed and compared across the groups. These measurements included CD4+, CD8+, CD3 + subsets, total lymphocytes, beta-2-microgloublin (beta2M), and neopterin. RESULTS: Significant differences were observed between the SBBC and the controls, particularly CD4% (p < 0.05), CD8 counts (p < 0.01), and CD4:CD8 ratios (p < 0.001). CONCLUSIONS: The results suggested that, as a group, the SBBC remained asymptomatic 12 to 16 years after infection with HIV-1. However, elevated CD8+ T lymphocytes, together with decreasing CD4%, suggested that some SBBC members were showing early immunologicalsigns of disease progression during late 1996, confirmed by recent (1998) follow-up studies.

Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1. A report from the Sydney Blood Bank Cohort.

BACKGROUND AND METHODS: The Sydney Blood Bank Cohort consists of a blood donor and eight transfusion recipients who were infected before 1985 with a strain of human immunodeficiency virus type 1 (HIV-1) with a deletion in the region in which the nef gene and the long terminal repeat overlap. Two recipients have died since 1994, at 77 and 83 years of age, of causes unrelated to HIV infection; one other recipient, who had systemic lupus erythematosus, died in 1987 at 22 years of age of causes possibly related to HIV. We present longitudinal immunologic and virologic data on the six surviving members and one deceased member of this cohort through September 30, 1998. RESULTS: The five surviving recipients remain asymptomatic 14 to 18 years after HIV-1 infection without any antiretroviral therapy; however, the donor commenced therapy in February 1999. In three recipients plasma concentrations of HIV-1 RNA are undetectable (<200 copies per milliliter), and in two of these three the CD4 lymphocyte counts have declined by 9 and 30 cells per cubic millimeter per year (P=0.3 and P=0.5, respectively). The donor and two other recipients have median plasma concentrations of HIV-1 RNA of 645 to 2850 copies per milliliter; the concentration has increased in the donor (P<0.001). The CD4 lymphocyte counts in these three cohort members have declined by 16 to 73 cells per cubic millimeter per year (P<0.001). In the recipient who died after 12 years of infection, the median plasma concentration of HIV-1 RNA was 1400 copies per milliliter, with a decline in CD4 lymphocyte counts of 17 cells per cubic millimeter per year (P=0.2). CONCLUSIONS: After prolonged infection with this attenuated strain of HIV-1, there is evidence of immunologic damage in three of the four subjects with detectable plasma HIV-1 RNA. The CD4 lymphocyte counts appear to be stable in the three subjects in whom plasma HIV-1 RNA remains undetectable.

IgA class antibodies and flow cytometric cross-matching in renal transplantation.

BACKGROUND: The established method of pretransplant cross-matching does not detect IgA antibodies, and IgA antibodies have thus been ignored when assessing patients for transplantation. The aim of this study was to detect IgA allo- and autoreactive antibodies using flow cytometry and to correlate the results with transplant outcome. METHODS: Pretransplant sera from 231 sequential renal recipients were tested for serum IgA levels and antibodies directed against the Fab portion of the human IgG molecule. Fifty-nine recipients with sufficient stored donor lymphocytes were also tested by flow cytometry for donor-specific alloantibodies of the IgA isotype. RESULTS: Graft survival was improved in recipients with higher IgA levels. High IgA anti-Fab levels led to a significantly higher 1-year graft survival (P<0.05). Graft survival was further enhanced where both serum IgA and IgA anti-Fab were raised (P<0.01). Although the mean IgA level tended to be higher for recipients with a positive IgA flow cytometric cross-match (FCXM), the IgA FCXM was not associated with increased IgA anti-Fab, suggesting that the IgA FCXM is detecting a different subset of IgA reactivity. Additionally, for primary grafts, a positive IgA FCXM was not associated with enhanced graft survival. CONCLUSIONS: Within the repertoire of IgA activity, there are two recognizable groups, the IgA anti-Fab specificity, which is significantly associated with enhanced graft survival, and that detected by the IgA FCXM, which surprisingly is more likely to be positive in less sensitized first grafts and is not associated with enhanced graft survival.

Strong human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte activity in Sydney Blood Bank Cohort patients infected with nef-defective HIV type 1.

Proposals for the use of live attenuated human immunodeficiency virus (HIV) type 1 (HIV-1) as a vaccine candidate in humans have been based on the protection afforded by attenuated simian immunodeficiency virus in the macaque model. Although it is not yet known if this strategy could succeed in humans, a study of the Sydney Blood Bank Cohort (SBBC), infected with an attenuated HIV-1 quasispecies with natural nef and nef/long terminal repeat deletions for up to 17 years, could provide insights into the long-term immunological consequences of living with an attenuated HIV-1 infection. In this study, HIV-specific cytoxic T-lymphocyte (CTL) responses in an SBBC donor and six recipients were examined over a 3-year period with enzyme-linked immunospot, tetrameric complex binding, direct CTL lysis, and CTL precursor level techniques. Strong HIV-specific CTL responses were detected in four of seven patients, including one patient with an undetectable viral load. Two of seven patients had weak CTL responses, and in one recipient, no HIV-specific CTLs were detected. High levels of circulating effector and memory HIV-specific CTLs can be maintained for prolonged periods in these patients despite very low viral loads.

Lymphoproliferative immune function in the Sydney Blood Bank Cohort, infected with natural nef/long terminal repeat mutants, and in other long-term survivors of transfusion-acquired HIV-1 infection.

OBJECTIVES: To assess T-helper cell immune function (proliferation) in members of the Sydney Blood Bank Cohort (SBBC) compared with other individuals with transfusion- and sexually acquired HIV-1 infection and with matched HIV-negative controls. DESIGN AND METHODS: Decreasing CD4 counts and T-helper cell function are associated with disease progression. Peripheral blood mononuclear cells (PBMC) from study subjects were assayed for in vitro proliferative responses to HIV-1-derived antigens, recall antigens and alloantigen. T-helper cell function and CD4 counts in members of the SBBC were followed longitudinally. RESULTS: Proliferative responses and CD4 counts from members of the SBBC were similar to or better than those of other transfusion- or sexually-acquired HIV-1-positive long-term non-progressors (LTNP), including the HIV-negative matched SBBC control groups. However, individuals with disease progression had reduced or undetectable proliferative responses to recall antigens but a conserved response to alloantigen; they also had low CD4 counts and low CD4:CD8 ratios. In the SBBC, these immune parameters were usually stable over time. CONCLUSIONS: The unique SBBC with natural nef/long terminal repeat deletions in the HIV-1 genome were genuine LTNP without showing signs of disease progression. They appeared to be a group distinct from the tail-end of the normal distribution of disease progression rates, and may remain asymptomatic indefinitely. The SBBC virus may form the basis of a live attenuated immunotherapeutic or immunoprophylactic HIV vaccine.

Donor helper T-cell frequencies as predictors of acute graft-versus-host disease in bone marrow transplantation between HLA-identical siblings.

BACKGROUND: Despite the current level of sophistication of molecular typing for class I and class II alleles, a significant proportion (20-40%) of recipients of HLA-identical sibling marrow develop severe, acute graft-versus-host disease (GVHD) after bone marrow transplantation. It has been suggested that the frequency of patient-specific helper T lymphocyte precursors (HTLp) detected in the HLA-identical sibling donor correlates with the incidence and severity of acute GVHD after transplantation. METHODS: This study group consisted of 42 patients who all received bone marrow from HLA-identical sibling donors from January 1990 to December 1996. Using a limiting dilution analysis, donor HTLp frequencies were determined on samples collected before transplantation. The HTLp assay used the cytotoxic T-cell line, CTLL-2, which proliferates in the presence of interleukin-2. The reliability and reproducibility of this assay was established by using cryopreserved batches of CTLL-2 cells of known sensitivity. RESULTS: The recipient-directed HTLp frequencies detected in the donor before transplantation were correlated with the incidence and severity of acute GVHD experienced by the recipient after transplantation. Statistical analysis revealed an extremely significant correlation between donor precursor frequencies and the development of acute GVHD in the patient after transplantation (P<0.0001). CONCLUSIONS: This study suggests that together with molecular typing the HTLp frequency should be considered when selecting the most suitable sibling donor for bone marrow transplantation.

A vaccine conjugate of 'ISCAR' immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil-based adjuvants.

TraT protein, known as ISCAR (= Immunostimulatory Carrier), is one of a family of integral membrane proteins (Imps) of Escherichia coli representing powerful carrier molecules which when injected into experimental animals generate substantial antibody and T proliferative responses to molecules conjugated to it. We extend these findings to show that ISCAR functions to stimulate Th1- and Th2-type responses, including specific cytotoxic T cells and tumour protection. We report here that by conjugating to ISCAR a 19mer peptide containing linear B epitopes, a T helper (Th) epitope, and a H-2b-restricted T cytotoxic (CTL) epitope of E7 protein of human papillomavirus type 16 (HPV16), and immunizing C57B1/6 (H-2b) mice, we elicited (i) specific IgG2a and IgG1 antibodies; (ii) IL-2 and IL-4 production by specifically recalled lymph node cells in vitro; (iii) cytotoxic T lymphocytes which specifically killed both E7 peptide-pulsed, and whole E7 gene-transfected tumour target cells; and (iv) in vivo protection against an E7 gene-transfected tumour cell inoculum. These findings have implications for the design of vaccines to stimulate immune responses to endogenously processed target antigens (e.g. tumour-associated antigens) without the unwanted side effects of oil-based adjuvants. In addition they support the case for a E7-targeted therapeutic vaccine for HPV-associated human cervical cancer.

Identification of the site of uptake of the E. coli heat-labile enterotoxin, LTB.

The results of this study demonstrate that the B subunit of the E. coli heat labile toxin (LTB) binds to the brush border of intestinal epithelial cells in a highly specific, lectin-like manner. Uptake of LTB and transcytosis to the basolateral side of the enterocytes can be observed within 1 h after feeding, and occurs through both the villous epithelial cells and the epithelial cells overlying lymphoid follicles and Peyer's patches. Binding and uptake most probably occur via receptor-mediated endocytosis, with GM1 ganglioside and galactoproteins on the enterocyte cell surface acting as specific ligands to which the LTB binds. Cell ELISA data, together with the observed distribution of immunocompetent cells and the localization of LTB binding, suggest that LTB which is taken up by the villous enterocytes enters the circulation and subsequently generates an IgG immune response in the spleen. At the same time, LTB which is taken up via the patch associated epithelium generates a local IgG and IgA immune response within the Peyer's patches and intestinal lymphoid follicles.

Peptide sequences with strong stimulatory activity for lymphoid cells: implications for vaccine development.

Seven peptides derived from the bacterial major outer-membrane protein TraT were synthesized and then tested in lymphoproliferative assays using lymphoid cells from a variety of animals that had been immunized with the native TraT molecule in saline. A hierarchical pattern of responsiveness to the peptides was observed in the four animal species studied and in particular three of the peptides (T2, T4 and T6) showed very strong responses in all species. The 'universality' of the TraT-derived peptides was confirmed by studying the responsiveness of lymphoid cells obtained from the peripheral blood of twenty clinically normal human donors. Thus, following a secondary in vitro immunization with TraT-pulsed human peripheral blood mononuclear cells, responsiveness to TraT and to the TraT-derived peptides was observed in the cultures derived from all twenty donors. Taken together, our findings imply that the putative T-cell epitope peptides (T2, T4 and T6) could be employed as carriers in subunit vaccines and thereby help to overcome the unresponsiveness observed in animals and humans as a result of MHC restriction.

Augmentation of HIV-specific lymphoproliferation in HIV-infected individuals by TraT: a novel T-cell immunopotentiating agent.

OBJECTIVE: To evaluate the potential of TraT to restore HIV-specific cell-mediated immunity. DESIGN: CD4+ T cell-associated antiviral and recall antigen-specific lymphoproliferative responses are generally impaired or absent in HIV-infected individuals. METHODS: Using peripheral blood mononuclear cells (PBMC) from a group of asymptomatic and symptomatic HIV-infected individuals, we compared the immunomodulatory effects of exogenous interleukin-2 (IL-2) with the effects elicited by the bacterial integral membrane protein, TraT. RESULTS: Exogenous IL-2 enhanced lymphoproliferation induced by an immunodominant synthetic HIV gp41 analogue, gp41[8] (amino acids 593-604), in four out of 10 asymptomatics and six out of 19 symptomatics. In contrast, TraT acted synergistically with gp41[8] to augment HIV-specific proliferation with higher frequency and greater magnitude than exogenous IL-2. Moreover, this TraT-mediated enhancement of HIV-specific lymphoproliferation occurred in the majority of HIV-infected individuals, irrespective of CD4+ T-cell count in peripheral blood or disease status, and thus appears not to be major histocompatibility complex-restricted. TraT also augmented lymphoproliferation induced by well-known recall antigens and other less immunodominant HIV analogues. CONCLUSIONS: These findings suggest that TraT, in combination with HIV-derived peptides, could be used to maintain or restore cell-mediated immune functions of HIV-infected individuals, as well as cellular immune functions in individuals suffering from other immunodeficiency disorders.