Recognition of heat shock protein 60 epitopes in children with type 1 diabetes.

BACKGROUND: Treatment with a specific HSP60 epitope in new onset of type 1 diabetes (T1D) patients has been shown to preserve endogenous insulin production. Previously, recognition of pan HLA-DR-binding HSP60 epitopes in various autoimmune diseases was found; this study investigated recognition of these epitopes in newly diagnosed T1D patients and correlated findings to the occurrence of a partial remission. METHODS: Peripheral blood mononuclear cells of 18 children with T1D were prospectively collected at disease onset and a few months after diagnosis. Epitope-specific T-cell proliferation and cytokine production (intracellular and in culture supernatants) were measured. Results were compared with 31 longstanding T1D patients and ten healthy controls. RESULTS: Although HSP60 epitope-specific T-cell proliferative responses were detected, overall proliferative responses were low. At onset, epitope-specific intracellular IFN-γ production was higher in T1D patients compared with healthy controls (p < 0.05). At follow-up, both IL-10 and IFN-γ production were higher in those without a partial remission than in those with a partial remission (both p < 0.05). Also, IL-10 and IFN-γ production were higher compared with onset for patients without a PR (both p < 0.01). In supernatants of HSP60 epitope-specific T-cell cultures, no substantial differences in cytokine production were found between T1D patients with and without a partial remission, either at onset or a few months after onset. As patient numbers were small, results should be interpreted with caution. CONCLUSIONS: Pan-DR-binding HSP60 peptides induced low peptide-specific proliferative responses and peptide-specific production of some, mainly intracellular, cytokines in T1D patients. Recognition did not differ significantly between patient groups and various time points.

Phenotypic and functional separation of memory and effector human CD8+ T cells.

Human CD8+ memory- and effector-type T cells are poorly defined. We show here that, next to a naive compartment, two discrete primed subpopulations can be found within the circulating human CD8+ T cell subset. First, CD45RA-CD45R0(+) cells are reminiscent of memory-type T cells in that they express elevated levels of CD95 (Fas) and the integrin family members CD11a, CD18, CD29, CD49d, and CD49e, compared to naive CD8+ T cells, and are able to secrete not only interleukin (IL) 2 but also interferon gamma, tumor necrosis factor alpha, and IL-4. This subset does not exert cytolytic activity without prior in vitro stimulation but does contain virus-specific cytotoxic T lymphocyte (CTL) precursors. A second primed population is characterized by CD45RA expression with concomitant absence of expression of the costimulatory molecules CD27 and CD28. The CD8+CD45RA+CD27- population contains T cells expressing high levels of CD11a, CD11b, CD18, and CD49d, whereas CD62L (L-selectin) is not expressed. These T cells do not secrete IL-2 or -4 but can produce IFN-gamma and TNF-alpha. In accordance with this finding, cells contained within this subpopulation depend for proliferation on exogenous growth factors such as IL-2 and -15. Interestingly, CD8+CD45RA+CD27- cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation. Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8+ T cell subset.

Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics.

To gain more insight into the role of HIV-1-specific cytotoxic T lymphocytes (CTL) in the pathogenesis of AIDS, we investigated temporal relations between HIV-1 Gag-specific precursor CTL (CTLp), HIV-1 viral load, CD4+ T cell counts, and T cell function. Six HIV-1-infected subjects, who were asymptomatic for more than 8 yr with CD4+ counts > 500 cells/mm3, were compared with six subjects who progressed to AIDS within 5 yr after HIV-1 seroconversion. In the long-term asymptomatics, persistent HIV-1 Gag-specific CTL responses and very low numbers of HIV-1-infected CD4+ T cells coincided with normal and stable CD4+ counts and preserved CD3 mAb-induced T cell reactivity for more than 8 yr. In five out of six rapid progressors Gag-specific CTLp were also detected. However, early in infection the number of circulating HIV-1-infected CD4+ T cells increased despite strong and mounting Gag-specific CTL responses. During subsequent clinical progression to AIDS, loss of Gag-specific CTLp coincided with precipitating CD4+ counts and severe deterioration of T cell function. The possible relationships of HIV-1 Gag-specific CTLp to disease progression are discussed.

Epstein-Barr virus-specific cytotoxic T cell responses in HIV-1 infection: different kinetics in patients progressing to opportunistic infection or non-Hodgkin's lymphoma.

Although the high incidence of EBV-associated diffuse large cell lymphomas (DLCL) in HIV-1 infection is believed to be related to loss of immune control due to HIV-induced immune deficiency, it has been claimed that cytotoxic T lymphocyte (CTL) responses to EBV are longer lasting in HIV-1-infected persons than CTL directed against HIV-1 itself. We approached this apparent paradox by performing the first longitudinal study into the kinetics of EBV and HIV-specific CTL responses in HIV-infected patients progressing either to AIDS with non-Hodgkin's lymphoma (NHL) or AIDS with opportunistic infection (OI). Multiple samples were tested from HIV-1 seroconversion to AIDS-diagnosis. Four out of six patients that were either long-term asymptomatic or progressing to OI showed declining HIV-1 CTL precursor (CTLp) frequencies whereas EBV-CTLp remained stable, suggestive for HIV-1-specific immune exhaustion. In two patients rapidly progressing to AIDS-OI, a parallel decline of HIV-1- and EBV-CTL responses was seen, indicative for total collapse of cellular immunity. In all these six patients EBV-load remained low. However, in four out of five patients that progressed to DLCL, EBV-load was high and increasing several months preceding the NHL. In all five patients, EBV-CTLp decreased before the emergence of the NHL. Thus, our data show that in HIV-1 infection loss of HIV-1-specific T cell immunity is not necessarily paralleled by loss of EBV-specific T cell responses. The occurrence of AIDS-related DLCL is preceded by decreasing EBV-CTLp and increasing EBV load. Failing EBV-control might therefore be an important step in the pathogenesis of AIDS-related DLCL.

Plasma granulysin levels and cellular interferon-gamma production correlate with curative host responses in tuberculosis, while plasma interferon-gamma levels correlate with tuberculosis disease activity in adults.

Granulysin is a recently identified cytolytic protein which is expressed by human cytotoxic T-lymphocytes and natural killer (NK)-cells, and has broad antimicrobial and tumoricidal activity. Circulating granulysin levels are associated with T- and NK-cell activity, and may thus reflect protection-associated cellular immune responses. In a case-control study in Indonesia, a highly tuberculosis (TB)-endemic country, we therefore determined plasma granulysin levels in adults with active pulmonary TB before, during, and after TB treatment, both in mild/moderate-TB and advanced-TB patients, and compared these to healthy neighbourhood controls. Adults with active pulmonary TB had significantly lower plasma granulysin levels compared to controls. After 2 months of anti-TB therapy, levels in TB patients had significantly increased, reaching values similar to those in controls. Plasma granulysin levels further increased after completion of TB therapy, being significantly higher than those in controls. Plasma granulysin levels correlated inversely with TB disease activity but not with TB disease severity. In contrast, plasma interferon-gamma (IFN-gamma) levels were significantly higher in active TB cases than in controls, normalised during treatment and correlated with both TB disease activity and TB disease severity. At the cellular level, granulysin and IFN-gamma expression both correlated inversely with disease activity. Interestingly, granulysin was predominantly expressed by IFN-gamma negative T-cells, suggesting that the cellular sources of IFN-gamma and granulysin in TB are partly non-overlapping. The observation that plasma granulysin levels and cellular IFN-gamma production correlate with curative host responses in pulmonary tuberculosis points to a potentially important role of granulysin, next to IFN-gamma, in host defence against M. tuberculosis.

Long-term survivors of HIV-1 infection.

The clinical course and outcome of HIV-1 infection are highly variable. A full spectrum of pathology has been observed, from rapid progression to AIDS within months of HIV-1 seroconversion, to asymptomatic survival for more than a decade. This phenomenon probably reflects the multiphasic and multifactorial nature of the virus-host interactions. Obviously, interest in the extremes now recognized in HIV-1 disease progression is growing, with the hope that mechanisms of protection may be found.

Selective in vitro expansion of HLA class I-restricted HIV-1 Gag-specific CD8+ T cells: cytotoxic T-lymphocyte epitopes and precursor frequencies.

OBJECTIVE: To identify HIV-1 Gag cytotoxic T-lymphocyte (CTL) epitopes and HLA restriction of their recognition, and to define precursor frequencies of HIV-1 Gag-specific CTL in the blood of seropositive individuals. METHODS: B-lymphoblastoid cell lines (B-LCL) infected with recombinant vaccinia viruses (rVV) containing a gene coding for HIV-1 Gag (rVV-Gag) were fixed with paraformaldehyde (PFA) and used as antigen-presenting cells (APC) to stimulate peripheral blood mononuclear cells (PBMC) from asymptomatic HIV-seropositive individuals. Specific CTL activity was determined in 51Cr-release assays using B-LCL as targets after infection with rVV-Gag or after pulsing with partially overlapping peptides spanning the Gag sequence. RESULTS: In vitro stimulation resulted in an increased number of CD8+ T cells and CD45R0+ and HLA-DR+ cells. Gag-specific cytotoxicity, mediated predominantly by HLA class I-restricted CD8+ CTL, was observed in all seven individuals studied. Multiple HLA-restricted CTL epitopes were identified with a single culture from one of the individuals. Gag-expressing APC were successfully used as stimulator cells in limiting dilution analysis to determine CTL precursor (CTLp) frequencies. CONCLUSION: PFA-fixed rVV-Gag-infected autologous B-LCL can be used as stimulator cells in bulk PBMC cultures to identify CTL epitopes and to determine CTLp frequencies. This method will facilitate the analysis of HIV-1-specific CTL responses in HIV-infected and vaccinated individuals.

Induction of a primary human cytotoxic T-lymphocyte response against a novel conserved epitope in a functional sequence of HIV-1 reverse transcriptase.

OBJECTIVE: To identify novel major histocompatibility complex (MHC) class I-restricted cytotoxic T-lymphocyte (CTL) epitopes conserved in HIV-1. METHODS: Potential conserved CTL epitopes were selected using a predictive computer algorithm based on a human leukocyte antigen (HLA)-A*0201 peptide-binding motif and tested for actual binding to the human processing defective cell line 174.CEM T2 (T2). Hence, the amino-acid sequences of 14 full-length sequenced HIV-1 strains were analysed. An in vitro primary peptide-specific human CTL response was induced with responding lymphocytes of an HIV-1-seronegative donor. Responding T cells were cloned by limiting dilution and tested for their ability to recognize naturally processed antigen in a 51Cr-release assay using recombinant vaccinia-HIV protein-infected B-lymphoblastoid cells (B-LCL) as target cells. RESULTS: The analysis of peptides bearing the HLA-A*0201 motif for conservation resulted in one peptide of Env, three of Gag and 12 of Pol. Only Gag340-348, Pol83-92, Pol267-277 and Pol960-968 showed binding properties to T2 comparable with those of known CTL epitopes Gag76-84 SLYNTVATL and Pol468-476 ILKEPVHGV. A successful primary MHC class I-restricted CTL response was induced against Pol468-476 and Pol267-277 VLDVGDAYFSV, a peptide in a functional sequence of reverse transcriptase (RT). The resulting CD8+ CTL clones were peptide-specific and able to specifically lyse recombinant vaccinia-HIV-1 RT-infected HLA-A*0201-matched B-LCL. CONCLUSION: The method used to screen proteins sequences for potential CTL epitopes, test selected peptides for binding to MHC class I and induction of an in vitro primary response against optimal binding peptides resulted in the identification of at least one novel conserved CTL epitope. The novel epitope is located in an area crucial for RT activity. This study demonstrates the feasibility of identifying highly conserved HIV-1-derived peptides capable of eliciting novel anti-HIV-1 CTL responses.

Immunopathology as a result of highly active antiretroviral therapy in HIV-1-infected patients.

OBJECTIVE: Unusual clinical inflammatory syndromes associated with underlying previously unrecognized opportunistic infections are increasingly being noted shortly after starting highly active antiretroviral therapy (HAART). This study examined the possible relationship between such unexpected disease manifestations and in vitro parameters of microbial antigen-specific immune reactivity in patients infected with HIV-1 who had a Mycobacterium avium intracellulare or Mycobacterium xenopi infection. DESIGN: In vitro T-cell proliferation experiments were performed after specific stimulation of a patient's peripheral blood mononuclear cells (PBMC) with M. avium and M. xenopi antigen and non-specific stimulation with phytohaemagglutinin (PHA). The results were compared with appropriate controls. PATIENTS: Five patients who presented with unusual clinical syndromes associated with M. avium or M. xenopi infection within weeks of experiencing large rises in CD4+ cell counts following the initiation of antiretroviral therapy. RESULTS: In all patients except one, mycobacteria-specific lymphoproliferative responses rose significantly following HAART; this was temporally associated with elevations in CD4+ cell counts and the occurrence of clinical disease. The patient with M. xenopi infection appeared to clear his infection subsequently without antimycobacterial therapy. In three of the four patients with M. avium infection, antimycobacterial treatment could be stopped without recurrence of infection. CONCLUSION: Our findings support the hypothesis that HAART may lead to clinically relevant inflammation as a result of restoration of specific immune reactivity against microbial pathogens that are subclinically present at the time treatment is initiated. Continuation of HAART may subsequently result in protective immunity and clearance of infection.

Decreased IFN- gamma and increased IL-4 production by human CD8(+) T cells in response to Mycobacterium tuberculosis in tuberculosis patients.

To investigate the role of MHC class I restricted CD8(+) T cells in host defense to M. tuberculosis, peripheral blood mononuclear cells (PBMC) from healthy BCG-vaccinated donors and untreated pulmonary tuberculosis (TB) patients in The Gambia were stimulated for 6 days with M. bovis BCG or M. tuberculosis and the CD8(+) T cell response analyzed. Intracellular FACS analysis of cytokine production by CD8(+) T cells showed that IFN- gamma and TNF- alpha production were greatly reduced in TB patients compared to healthy controls. IL-4-producing CD8(+) T cells were detected in TB patients, a phenotype absent in controls. Collectively, these data suggest that an alteration in the type 1/type 2 cytokine balance occurs in CD8(+) T cells during clinical tuberculosis, and that this may provide a surrogate marker for disease.

Kinetics of immune functions and virus replication during HIV-1 infection.

INTRODUCTION: Kinetics of human immunodeficiency virus type 1 (HIV-1) cytotoxic T lymphocyte (CTL) responses and viral load were evaluated in HIV-1 infected homosexual men who progressed to AIDS within 3-6 years after seroconversion and in long-term survivors who remained AIDS-free for > 9 years with normal CD4+ T cell counts. METHODS: CTL against four major HIV-1 gene products (i.e. Gag, reverse transcriptase (RT), Nef and Env) were expanded in vitro under limiting dilution conditions using antigen specific stimulation. CTL activity was measured in standard split-well 51Cr-release assay. Viral load was measured both as serum HIV-1 RNA levels and frequency of circulating CD4+ T cells productively infected with HIV-1. Polyclonal T cell function in vitro was determined in whole blood lymphocyte cultures, measuring lymphoproliferative responses to CD3 monoclonal antibody. RESULTS: Long-term survival was associated with either persistently high or stable low HIV-1 specific CTL responses, accompanied by preserved in vitro polyclonal T cell reactivity and low viral load. In progressors, HIV-1 specific CTL responses were initially generated with similar kinetics as compared to long-term survivors. However, with progression to AIDS antiviral CTL activity and T cell function deteriorated simultaneously, while viral load increased. CONCLUSIONS: Our results are consistent with the hypothesis that HIV-1 specific CTL are beneficial through control of viremia to the virologic set-point and contribute to maintenance of the asymptomatic phase. However, loss of HIV-1 specific immune control as part of a more general loss of T cell function is the precipitating event in AIDS pathogenesis.

Antigen-specific T-lymphocyte proliferative responses during highly active antiretroviral therapy (HAART) of HIV-1 infection.

To evaluate functional T-cell recovery during combination therapy with ritonavir, lamivudine (3TC), and zidovudine (ZDV), peripheral blood mononuclear cells (PBMC) were obtained from 4 HIV-1 infected patients (baseline values: 40 403 CD4+ T-cells/microl; 4.6-6.4 log HIV-1 RNA copies/ml) before HAART administration (week -1) and after 5, 20, and 37 weeks of treatment on average. In vitro lymphoproliferative responses (LPR) to C. albicans, tetanus toxoid, and M. tuberculosis protein purified derivative (PPD), as recall antigens (Ag), and to recombinant HIV-1 Gag-p24 and p17 were measured by 3H-Thymidine incorporation. LPR to recall Ag, almost undetectable before therapy, appeared in all four patients during HAART soon after maximal load reduction was achieved. LPR to Gag-p17, but not to p24, became also detectable in three patients, even though remaining weak. In conclusion, improved T-lymphocyte function during HAART was achieved probably mostly as a result of lower virus inhibitory factors and cytokines.

Gag-specific immune responses after immunization with p17/p24:Ty virus-like particles in HIV type 1-seropositive individuals.

Gag-specific immune responses and changes in HIV-1 RNA levels were evaluated in eight HIV-1-infected persons, in order to assess the immunotherapeutic potential HIV-1 p17/p24: Ty virus-like particles (p24-VLP). All treated subjects showed transient and dose-dependent proliferative responses to the Ty-VLP carrier (stimulation index [SI], 2.0-119.5). Three of four individuals who received either 500 or 1,000 micrograms of p24-VLP also showed proliferative responses to p17 or p24 (SI, 2.0-15.7). In 2 subjects who were treated with either 500 or 1,000 micrograms of p24-VLP, enhanced Gag-specific CTL precursor (CTLp) frequencies were observed after immunization (10- to 14-fold). Both subjects had low baseline Gag-specific CTL activity (< 25 cTLp/10(6) PBMCs). In the other participants studied no significant boosting of preexisting Gag-specific CTL responses was observed. Short-term elevation of HIV-1 RNA levels at weeks 2 and 4 was observed in two subjects treated with the highest dose of p24-VLP. However, HIV-1 RNA levels at week 24 did not significantly differ from those found in the placebo group. In conclusion, p24-VLP induced marginal Gag-specific immune responses in limited numbers of HIV-1-seropositive individuals, with some showing transient elevation of HIV-1 viral load. Further studies are needed to establish potential clinical effects of these observations.

Molecular analysis of critical sequences within the EBNA-2 type 1 gene from Epstein-Barr virus isolates from patients with infectious mononucleosis, tonsillar hyperplasia, and HIV infection.

EBNA-2 is the first protein to be detected after infection of primary B lymphocytes by Epstein-Barr virus (EBV) and plays an essential role as transcriptional activator in EBV-induced lymphocyte transformation. We analysed by PCR and sequencing regions of the EBNA-2 type 1 gene from isolates from 13 children with infectious mononucleosis (IM), 6 children with tonsillar hyperplasia (TH), and 9 patients with HIV infection followed longitudinally. We found in all three groups of patients frequent non-silent point mutations at positions 48990, 48991, 49021, 49057, 49083, 49089, 49091, 49113, 49119, 49140, 49156, and a triplet insertion at position 49136. While 4 out of 13 samples from patients with IM showed a mosaic pattern suggesting co-existence of more than 1 substrain of EBNA-2 type 1, none of the samples from TH showed this pattern consistent with substrain selection during clinical latency. No sequence changes were noted over time in samples derived from patients with HIV infection. We conclude that in analogy to the coexistence of several subtypes of EBNA-1 in healthy EBV carriers, samples from IM can harbor more than one subtype of the EBNA-2 type 1 gene.

Mycobacterium-specific human CD8 T cell responses.

Tuberculosis (TB) remains a global health problem. There is an intense effort to identify correlates of protective immunity and to design new TB vaccines. CD8 T cells are thought to play a significant role in controlling Mycobacterium tuberculosis infection. Relatively little has been published about the antigens and epitopes targeted by mycobacteria-specific CD8 T cells. Here we present an update of our 1999 overview of human CD8 T cell epitopes in mycobacterial antigens and discuss related issues relevant to TB diagnosis and vaccine development.

CD8+ T lymphocytes against mycobacterium tuberculosis.

Tuberculosis (TB) is again a global health problem. Identification and characterization of the correlates of protective immunity against TB is critical for the rationale design of novel TB vaccines. There is accumulating data that CD8+ T lymphocytes are involved in the immune response against mycobacteria. Here the current state of the art is reviewed with respect to phenotype, specificity and effector mechanisms of mycobacteria-specific CD8+ T lymphocytes. In addition, the first listing is presented of mycobacteria-derived CD8+ cytotoxic T lymphocyte (CTL) epitopes containing sequences published up to the end of 1998.