A virus-sensitive suppressor cell is involved in the regulation of human allospecific T cell-mediated cytotoxicity

The in vitro generation of allospecific CTL by human PBMC was enhanced 4- to 16-fold by sequential plastic and nylon wool adherence, which depleted the PBMC of macrophages and B cells. The enhanced CTL response was suppressed by adding back irradiated, unfractionated PBMC or adherent cells to the depleted cells. This finding suggests that the enhanced CTL response was not simply a consequence of enrichment of T cells, but was instead due to active suppression by radioresistant cells contained in the adherent fraction. Of note is the finding that, unlike the CTL response, the proliferative response to allostimulation was not affected by the removal of adherent cells. The suppressor function could be abrogated by preincubation of irradiated PBMC with influenza A virus before the coculture with depleted cells. Furthermore, costimulation of unfractionated PBMC with influenza A virus and allogeneic stimulators augmented allospecific CTL activity. Thus, in the adherent fraction of human PBMC, there appears to be a native suppressor population that can be functionally inactivated by virus. This result may account for the clinical observation of increased allograft rejection after certain viral infections.     

Immunological properties of umbilical cord blood-derived mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are promising candidates for developing cell therapies for intractable diseases. To assess the feasibility of transplantation with human umbilical cord blood (hUCB)-derived MSCs, we analyzed the ability of these cells to function as alloantigen-presenting cells (APC) in vitro. hUCB-MSCs were strongly positive for MSC-related antigens and stained positively for human leukocyte antigen (HLA)-AB and negatively for HLA-DR. When treated with interferon (IFN)-γ, the expression of HLA-AB and HLA-DR, but not the co-stimulatory molecules CD80 and CD86, was increased. hUCB-MSCs did not provoke allogeneic PBMC (peripheral blood mononuclear cell) proliferation, even when their HLA-molecule expression was up-regulated by IFN-γ pretreatment. When added to a mixed lymphocyte reaction (MLR), hUCB-MSCs actively suppressed the allogeneic proliferation of the responder lymphocytes. This suppressive effect was mediated by soluble factors. We conclude that hUCB-MSCs can suppress the allogeneic response of lymphocytes and may thus be useful in allogeneic cell therapies.     

Antigen presentation by interferon-gamma-treated endothelial cells and fibroblasts: differential ability to function as antigen-presenting cells despite comparable Ia expression

The effect of interferon-gamma (IFN-gamma) on endothelial cell (EC) and fibroblast (FB) class II major histocompatibility complex (MHC) gene product expression and antigen presenting ability was examined. Control FB did not express class II MHC gene products, whereas a small (less than 1%) population of passaged EC expressed class II gene products. IFN-gamma induced a comparable density of HLA-DR expression on nearly all EC and FB. IFN-gamma-treated EC and FB also expressed HLA-DP but at a lower density, whereas HLA-DQ expression was barely detectable on either cell type. Control FB were not able to stimulate allogeneic T4 cell DNA synthesis or function as antigen-presenting cells (APC). Control EC were also unable to stimulate allogeneic T4 cell DNA synthesis unless large numbers of stimulator cells were used. Small numbers of IFN-gamma-treated EC were able to stimulate allogeneic T4 cell DNA synthesis, whereas larger numbers were markedly more effective than control EC. In contrast, IFN-gamma-treated FB were ineffective stimulators of allogeneic T4 cell DNA synthesis. IFN-gamma-treated FB were able to present the exogenous antigen SKSD to autologous but not allogeneic T4 cells, but they were extremely inefficient APC. The inability of IFN-gamma-treated FB to function as APC could not be explained by FB-mediated immunosuppression, Ia density, or HLA-DQ expression. This limited capacity of IFN-gamma-treated FB to participate in Ia-restricted functional interactions with T4 cells correlated with a similar diminished capacity to support nonspecific mitogen-induced proliferation of T4 cells before IFN-gamma-induced Ia expression. This accessory cell function was not enhanced by IFN-gamma treatment. Monocytes syngeneic to the responding T4 cells but not interleukin 1 (IL 1) permitted IFN-gamma-treated FB but not control FB to stimulate allogeneic T4 cell DNA synthesis, but they remained markedly less effective stimulators than monocytes. Moreover, IFN-gamma-treated FB were effective stimulators of alloprimed T4 cells, in contrast to their inability to stimulate fresh T4 cells. Furthermore, monocytes and IFN-gamma-treated FB were comparably effective stimulators of alloreactive T cell lines. These data suggest that accessory cells perform functions unrelated to Ia and IL 1 that are necessary for mitogen-, alloantigen-, and antigen-induced proliferation of freshly isolated T cells. Monocytes and EC effectively perform this function, but FB do not. This accessory cell function does not seem to be as important for the activation of primed T cells.     

T cell recognition of allopeptides in context of syngeneic MHC.

We have analyzed the ability of T cells to recognize peptides corresponding in sequence to an allogeneic HLA-DR molecule, in context of syngeneic MHC. PBMC from a responder with the HLA-DR beta 1*1101/DR beta 1*1201 genotype were stimulated in vitro with a mixture of four synthetic peptides derived from the first domain of the DR beta 1*0101 chain (amino acid residue 1-20, 21-42, 43-62, and 66-90). An alloreactive T cell line, TCL-LS, which proliferates only in response to peptide 21-42 presented by HLA-DR beta 1*1101, was obtained. The blastogenic response of the line was inhibited by anti-HLA-DR and CD4 antibodies but was not affected by antibodies to HLA-DQ, HLA-DP, HLA-ABC, and CD8. In the presence of irradiated, autologous APC, TCL-LS displayed specific proliferative responses to stimulating cells obtained from individuals carrying the DR beta 1*0101 allele. In the absence of autologous APC, TCL-LS recognized HLA-DR1 on allogeneic cells only when expressed together with HLA-DR beta 1*1101, the restrictive element. This indicates that TCL-LS recognizes processed HLA-DR1 molecule presented as nominal Ag. Study of TCR-V beta gene repertoire expressed by TCL-LS showed that only two V beta genes were used (V beta 13.2 and V beta 12). Two T cell clones (TCC) derived from this line, TCC-A5 and B4, exhibited a similar pattern of reactivity and expressed V beta 13.2. These results indicate that T cells recognizing peptides, which are derived from the breakdown of allogeneic MHC class II proteins and are presented by self-HLA-DR molecules, participate in allorecognition.     

Anti-apoptotic activity of the glutathione peroxidase homologue encoded by HIV-1

The third reading frame of the envelope gene from HIV-1 codes for a protein homologous to the human selenoprotein glutathione peroxidase (GPX). Cells stably or transiently transfected with a HIV-1 GPX construct are protected against the loss of the mitochondrial transmembrane potential and subsequent cell death induced by exogenous reactive oxygen species (ROS) as well as mitochondrion-generated ROS. However, HIV-1 GPX does not confer a general apoptosis resistance, because HIV-1 GPX-transfected cells were not protected against cell death induced by staurosporine or oligomycin. The inhibition of cell death induced by the ROS donor tert-butylhydroperoxide was also observed in cells depleted from endogenous glutathione (GSH), suggesting that GSH is not the sole electron acceptor for HIV-1 GPX. Clinical HIV-1 isolates from long-term non-progressors (untreated patients with diagnosed HIV-1 infection for > 10 years, with CD4 T cell count of > 500 cells/mm3) mostly possess an intact GPX gene (with only 18% of loss-of-function mutations), while HIV-1 isolates from patients developing AIDS contain non-functional GPX mutants in 9 out of 17 cases (53%). Altogether, these data suggest that HIV-1 GPX possesses a cytoprotective, pathophysiologically relevant function.     

CD4+ T cell responses elicited by different subsets of human skin migratory dendritic cells

Skin dendritic cells (DC) are professional APC critical for initiation and control of adaptive immunity. In the present work we have analyzed the CD4+ T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin explants, including CD1a+CD14- Langerhans' cells (LC), CD1a-CD14- dermal DC (DDC), and CD1a-CD14+ LC precursors. Skin migratory DC consisted of APC at different stages of maturation-activation that produced IL-10, TGF-beta1, IL-23p19, and IL-12p40, but did not release IL-12p70 even after exposure to DC1-driving stimuli. LC and DDC migrated as mature/activated APC able to stimulate allogeneic naive CD4+ T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4+ T cell stimulatory function of LC and DDC correlated with their high levels of expression of MHC class II, adhesion, and costimulatory molecules. The Th1-biasing function of LC and DDC depended on their ability to produce IL-23. By contrast, CD1a-CD14+ LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4+ T cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function of CD14+ LC precursors increased significantly by augmenting their cell number, prolonging the time of interaction with responding T cells, or addition of recombinant human IL-23 in MLC. The data presented in this study provide insight into the function of the complex network of skin-resident DC that migrate out of the epidermis and dermis after cutaneous immunizations, pathogen infections, or allograft transplantation.     

In vitro expansion of Ag-specific T cells by HLA-A*0201-transfected K562 cells for immune monitoring

BACKGROUND: Development of a practical and sensitive assay for evaluating immune responses against cancer Ag has been a challenge for immune monitoring of patients. We have established a reproducible method using peptide-pulsed K562-A*0201 cells as APC to expand Ag-specific T cells in vitro. This method may be applied for monitoring T-cell responses in cancer immunotherapy clinical trials. METHODS: Autologous PBMC from HLA-A*0201+ healthy donors and patients with melanoma were stimulated with peptide-pulsed K562-A*0201 cells under varying conditions. We investigated (1) different culture conditions, including the requirements for serum and cytokines for expansion of CD8+ T lymphocytes; (2) a range of peptide concentrations for Ag loading; (3) phenotypic characterization of responding T cells; and (4) APC:responder ratios and their effects on T-cell expansion. We validated these conditions by ELISPOT and intracellular cytokine staining (ICS) assays using peptides from influenza, Epslein-Barr Virus (EBV) and tyrosinase. RESULTS: Conditions for optimal T-cell expansion using K562-A*0201 APC included input of 2 x 10(6) PBMC, a 10 microg/mL peptide concentration to pulse K562-A*0201 cells, a 1:30 APC:responder T-cell ratio and culture in 10% autologous plasma supplemented with IL-2 and IL-15. In these conditions, Ag-specific T cells expanded >100-fold over a 10-day culture period (peak at day 12). DISCUSSION: This bulk culture method is simple and reliable for expanding human Ag-specific T cells using peptide-pulsed K562-A*0201 cells. This HLA-matched APC line can be adapted to other HLA haplotypes, and has advantages for monitoring clinical trials of immunotherapy with limited availability of autologous APC and PBMC from patients.     

Inhibition of stimulation in murine mixed lymphocyte cultures with an alloantiserum directed against a shared Ia determinant.

Pools of high titered alloantisera were raised by immunizing (B10.A/SgSn X A/WySn)F1 mice with C57BL/10Sn(B10) spleen cells. This serum (F1 anti-B10), when added to one-way mixed lymphocyte cultures (MLC), inhibited stimulation of B10.A splenic responders by both B10 and B10.D2/nSn irradiated, splenic stimulators. The B10 stimulation was suppressed approximately 85% whereas the mean suppression of B10.D2 stimulation was approximately 60%. In the ofrmer case, the serum contained antibodies reactive with multiple major histocompatibility complex determinants on the stimulator cells. In the latter case, the cytoxic reactivity of the serum was directed principally against an I region-associated determinant Ia.8) shared by B10 and B10.D2 and coded for by a gene(s) in the I-A subregion. The magnitude of the suppression of the response to B10.D2 cells (60%) was similar to the reduction in stimulation observed when the Ia.8 difference was eliminated genetically by using (B10 X B10.A)F1 responder cells against irradiated B10.D2 stimulators. Ihhibition of MLC by this antiserum was a function of reactivity with stimulator and not responder cells. Although some pools of F1 anti-B10 antiserum produced partial inhibition of the responder cell in a B10.D2 vs B10.Ax MLC combination, the results were inconsistent and not correlated with the anti-Ia.8 cytotoxicity titers. In addition, an F1 anti-B10 antiserum pool, which consistently failed to inhibit the responder cell, nevertheless inhibited both irradiated B10.D2 and (B10.A X B10.D2)F1 cells from stimulating B10.A responder cells. However, this same antiserum did not inhibit stimulation of B10.D2 responder cells by the (B10.A X B10.D2)F1 stimulators. Thus, the binding of antibodies to the non-stimulating antigens on the F1 stimulator cell did not interfere with the capacity of the appropriate stimulating antigens to cause stimulation. All of these results are consistent with the hypothesis that Ia allo-antigens are the major stimulating determinants of I region-associated MLC reactions.     

Non-H-2-linked genetic regulation of cytotoxic responses to hapten-modified syngeneic cells. I. Non-H-2-linked Ir gene defect expressed on T cells is not predetermined at the stage of bone marrow cells

Spleen cells from C3H/He or BALB.K mice immunized to the newly synthesized amino-reactive hapten 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide ester (AED-NH2) were stimulated in vitro with AED-NH2-modified syngeneic cells. After 5 days of culture, effector cells were assayed for their cytotoxic activity against AED-NH2-modified target blast cells. C3H/He and BALB.K mice exhibited the respective high and low anti-AED-NH2 cytotoxic T lymphocyte (CTL) responses. This contrasted with the observation that both of these H-2k strains generated potent CTL responses against aminoreactive haptens, e.g., trinitrophenyl (TNP). Because C3H.SW and BALB.B strains, which are the H-2b counterpart of the above two strains, also represented the respective high and low responders to AED-NH2 hapten, this hapten model enabled us to investigate cellular mechanisms underlying the above non-H-2-associated genetic regulation of CTL responses (C3H vs BALB non-H-2 backgrounds). The results demonstrated that there was no detectable difference between C3H/He and BALB.K strains in the lysability of target cells and the ability of stimulating cells to activate primed spleen cells. Anti-AED-NH2 CTL responses were only marginal when antigen-presenting cells (APC) were eliminated from the primed spleen cells of high responder C3H/He or (C3H/He X BALB.K)F1 mice. The addition of APC to cultures free of APC regained an appreciable CTL response in C3H/He or (C3H/He X BALB.K)F1 mice, irrespective of whether APC were derived from high (C3H/He) or low (BALB.K) responders. We have also demonstrated that allogeneic radiation bone marrow chimera (BALB.K----C3H/He) exhibited a CTL response comparable to that induced by C3H/He mice, whereas the reverse direction of allogeneic chimera (C3H/He----BALB.K) induced a marginal CTL response. These results indicate that this non-H-2-associated Ir gene defect is expressed on T cells (CTL precursors and/or helper T cells) rather than APC, and that this T cell defect is not predetermined at the level of bone marrow cells. The results are discussed in the light of the genetic and cellular mechanisms underlying non-H-2-linked Ir gene control.     

A model for the selective loss of major histocompatibility complex self-restricted T cell immune responses during the development of acquired immune deficiency syndrome (AIDS)

Functional analyses of peripheral blood leukocytes (PBL) from high risk HTLV-III antibody-negative and antibody-positive donors, as well as from patients with acquired immune deficiency syndrome (AIDS), lymphadenopathy syndrome (LAS), and AIDS-related complex (ARC) were performed by the in vitro generation of cytotoxic T lymphocyte (CTL) and proliferative responses to the HLA self-restricted antigens of influenza virus (S + X) and to nonself restricted HLA alloantigens (ALLO). All 40 antibody-negative donors tested responded to both S + X and ALLO in the CTL response, whereas six of 14 antibody-positive, two of three LAS, four of five ARC, and seven of 17 AIDS patients exhibited a selective absence of CTL to S + X, but generated normal or elevated CTL responses to ALLO. Of the remaining 10 AIDS patients, nine did not respond to either S + X or ALLO, and one responded to both S + X and ALLO. A similar selective loss of the proliferative response to S + X was found. We also observed antibody-positive donors who initially generated CTL responses to S + X and ALLO, but lost the S + X response as a function of time. We were able to restore the selective loss of S + X CTL activity in vitro by the addition of IL 2 and, to some extent, by co-stimulation with S + X plus ALLO. Depletion of CD4+ T helper cells and removal of autologous antigen-presenting cells from the PBL of healthy antibody-negative donors indicated that distinct T helper cell subsets exist in human PBL, and that S + X responses must use a CD4+ T helper population, whereas ALLO responses can utilize an alternate CD4- T helper pathway. A model is presented indicating the selective depletion of CD4+ T helper function in the developmental stages of AIDS. The functional test for T helper activity to self restricted antigens may be the earliest indicator of immune functional loss in the development of AIDS, and may precede a reduction in the absolute number of CD4+ cells.     

Differential sensitivity of human T helper cell pathways by in vitro exposure to cyclosporin A

Cyclosporin A (CsA) is a widely used agent for the prevention of tissue allograft rejection in human transplantation. As a result of the recent demonstration that the allospecific Th cell response of human PBL can be generated by three distinct pathways of Th cell and APC interactions, we investigated the sensitivity of these three Th-APC pathways, as well as the Th response to recall Ag, to different concentrations of CsA. PBL from healthy volunteer donors were set up as primary in vitro cultures either without antigenic stimulation, or with influenza A virus, tetanus toxoid, or HLA alloantigenic (ALLO) stimulation. Ag-stimulated IL-2 production and proliferation were measured to assess Th cell function. To study the effect of CsA on Th function, different concentrations of CsA (0.001 to 0.1 micrograms/ml final) were added to the cultures at the time of stimulation. Th responses to influenza A virus and tetanus toxoid were more sensitive to CsA than the Th response to ALLO. By selective depletion of either responder or stimulator APC and/or of CD4+ or CD8+ cells, we 1) verified that the human ALLO Th response can be mediated by three distinct Th-APC pathways; 2) demonstrated that the ALLO response mediated by CD4+ Th and self-APC (the same helper pathway used by recall Ag) is as sensitive to CsA as the responses to recall Ag; and 3) showed that there is a hierarchy of sensitivity of these three allospecific pathways. The results are discussed with respect to the potential significance of the differential sensitivity of these allospecific Th-APC pathways to CsA for prevention of tissue allograft rejection.     

Critical role of nitric oxide during the apoptosis of peripheral blood leukocytes from patients with AIDS

BACKGROUND: Highly active antiretroviral therapies (HAART) increase the CD4(+) cell count, but complete normalization of this parameter has not been obtained in some patients. As oxidative stress plays an important role during human immunodeficiency virus type 1 (HIV-1)-associated dementia and lymphocyte apoptosis, we asked whether the nitric oxide (NO) pathway plays a role in the in vitro survival of peripheral blood mononuclear cells (PBMC) from HIV-1(+) patients and how it correlates with peripheral CD4(+) cell levels. MATERIALS AND METHODS: PBMC were isolated from patients with AIDS and assayed for apoptosis and proliferation in the presence of various chemicals, including agonists or antagonists of the NO pathway. Data were then compared with several in vivo parameters from the same patients. RESULTS: Apoptosis of PBMC in the presence of exogenous NO is significantly higher in patients with low peripheral CD4(+) cell levels than in patients with high CD4(+) cell numbers or seronegative individuals. In addition, endogenous NO inhibition rescues cells from apoptosis in AIDS patients with low circulating CD4(+) cell numbers and helps recovery of the T cell proliferative response. NO-mediated apoptosis does not require cGMP but involves peroxynitrite generation, PARP activation, and NAD(+) depletion. CONCLUSIONS: Taken together, the data suggest the involvement of NO during the apoptosis and functional impairment of lymphocytes in patients with AIDS.     

Similar replication capacities of primary human immunodeficiency virus type 1 isolates derived from a wide range of clinical sources.

Numerous studies have suggested that there are significant differences in replication capacities and cytopathicities among human immunodeficiency virus type 1 (HIV-1) isolates and that these differences correlate with the clinical status and geographical origin of infected individuals. However, it has been difficult to assess whether reported distinctions could be attributed to the methods used or whether they imply a true disparity between viral isolates. We thus attempted to characterize the replication properties of HIV-1 isolates directly recovered from infected patients (primary isolates) by using a standardized infection assay. Viruses were isolated from patients' peripheral blood mononuclear cells (PBMC) by a single coculture with normal donor PBMC stimulated with phytohemagglutinin. Replication curves and cytopathic effect of a standard inoculum (1 ng of p24) of 66 primary HIV-1 isolates were similar regardless of clinical stage of the patient (asymptomatic, AIDS-related complex, or AIDS) and evolutive feature (rate of progression to AIDS). There was no difference between viruses derived from patients sensitive to zidovudine and those derived from patients resistant to zidovudine. Moreover, no difference was found among viral isolates of different geographical origins (Central Africa, Zaire, Brazil, or France). Similarly, the replication patterns and cytopathicities of isolates from bronchoalveolar lymphocytes did not differ from those of isolates derived from PBMC. In contrast, the same amount of viral inoculum of five laboratory HIV-1 strains (HIV-1, EL1, SF, MN, and RF) produced different replication curves and were much less cytopathic. In contrast to laboratory viral strains, it appears that the primary HIV-1 isolates tested, whatever their clinical status and source, exhibited similar replication capacities and cytopathicities in allogeneic donor PBMC.     

Cooperation between CD4 T helper cells is required for the generation of alloantigen-specific, IFN-gamma-producing human CD4 T cells

We have used MLR to assess the cell interactions that enable human PBL stimulated with irradiated, allogeneic PBL to give rise to allospecific, IFN-gamma-producing CD4 T cells. We were able to generate alloantigen-specific, IFN-gamma-producing T cells from peripheral blood. The responder and stimulator cell numbers required for the optimal generation of such cells, enumerated using an enzyme-linked immunospot assay, were separately determined for various combinations of responders and stimulators. The number of antigen-specific, IFN-gamma-producing cells generated per 10(6) responder cells, seeded at the initiation of culture, is critically dependent on the density of the responder cells, with minimal generation of IFN-gamma-producing T cells at low responder densities. These and further observations with fractionated responding PBL show that CD4 T-cell/CD4 T-cell interactions, which are completely independent of CD8() T cells, are necessary for the in vitro generation of alloantigen-specific, IFN-gamma-producing CD4 T cells.     

Human epidermal cells are more potent than peripheral blood mononuclear cells for the detection of weak allogeneic or virus-specific primary responses in vitro

Human epidermal cells (EC) and peripheral blood mononuclear cells (PBMC) have been used as antigen-presenting cells in allogeneic reactions or in self-restricted antiviral responses. Comparison of results from both cell types indicates that: (1) EC were better stimulators of primary proliferative responses in all the antigenic systems tested. (2) In secondary reactions, EC and PBMC functioned similarly for allogeneic responses, while a weak but significant difference could be observed in both (HSV1 or influenza A) virus-specific reactions. (3) By comparing pairs of HLA-identical mixed lymphocyte reaction (MLR)-negative siblings, positive responses were observed in several different families when lymphocytes of potential bone marrow donors were stimulated by EC of the recipient. This suggests that EC might be useful in detecting relatively weak proliferative responses in a number of antigenic systems, but especially in primary reactions against viral or putative minor histocompatibility antigens. (4) Despite this stronger antigen-presenting capacity in proliferative responses, EC induced lower levels of cytotoxic T lymphocyte (CTL) reactions than PBMC, not only in allogeneic responses but also in virus-specific self-restricted reactions.     

Quantitation of human immunodeficiency virus type 1 DNA forms with the second template switch in peripheral blood cells predicts disease progression independently of plasma RNA load

There are several forms of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood T cells and lymph nodes in untreated HIV-1-infected individuals and in patients whose plasma HIV-1 RNA levels are suppressed by long-term combination antiretroviral therapy. However, it remains to be established whether the concentration of HIV-1 DNA in cells predicts the clinical outcome of HIV-1 infection. In this report, we measured the concentration of HIV-1 DNA forms which has undergone the second template switch (STS DNA) and 2-long-terminal-repeat DNA circles in peripheral blood mononuclear cell (PBMC) samples. To do this, we used molecular-beacon-based real-time PCR assays and studied 130 patients with hemophilia in the Multicenter Hemophilia Cohort Study. We assessed the influence of baseline HIV-1 STS DNA levels on the progression of HIV-1 disease in the absence of combination antiretroviral therapy by Kaplan-Meier and Cox regression analysis. Among the patients who progressed to AIDS, the median levels (interquartile ranges) of STS HIV-1 DNA in PBMC were significantly higher than those of patients who remained AIDS free during the 16 years of follow-up (1,017 [235 to 6,059] and 286 [31 to 732] copies per 10(6) PBMC, respectively; P < 0.0001). Rates of progression to death and development of AIDS varied significantly (log rank P < 0.001) by quartile distribution of HIV-1 STS DNA levels. After adjustment for age at seroconversion, baseline CD4(+) T-cell counts, plasma viral load, and T-cell-receptor excision circles, the relative hazards (RH) of death and AIDS were significantly increased with higher HIV-1 STS DNA levels (adjusted RH, 1.84 [95% confidence interval (CI), 1.30 to 2.59] and 2.62 [95% CI, 1.75 to 3.93] per 10-fold increase per 10(6) PBMC, respectively). HIV-1 STS DNA levels in each individual remained steady in longitudinal PBMC samples during 16 years of follow-up. Our findings show that the concentration of HIV-1 STS DNA in PBMC complements the HIV-1 RNA load in plasma in predicting the clinical outcome of HIV-1 disease. This parameter may have important implications for understanding the virological response to combination antiretroviral therapy.     

H-2K/H-2D and Mls and I region-associated antigens stimulate helper factor(s) involved in the generation of cytotoxic T lymphocytes

This study examines the antigen that stimulate production or release of a soluble helper factor(s) involved in development of cytotoxic T lymphocytes (CTL). Antigens associated with the Mls locus, I and K/D regions of the MHC were all capable of stimulating responder cells in MLC to produce helper factor. These supernatant fluids were all capable of providing "help" for the generation of cytotoxic T lymphocytes in MLC in which spleen cells are stimulated by allogeneic heat-treated thymocytes or splenocytes. Previous reports from our laboratory as well as others have shown that heat-treated cells do not stimulate a cytotoxic response. Heat-treatment of Mls, I, and H-2K/H-2D region incompatible stimulatory cells in MLC eliminated their ability to induce responder cells to produce helper factor, suggesting this is the mechanism whereby heat-treatment reduces the ability of cells to stimulate cell-mediated lympholysis (CML). The inability of supernatant fluids, from MLCs in which heat-treated cells were the stimulators, to assist in the generation of cytotoxic T cells did not appear to be the result of any suppressive factor induced by such treatment. Further, the antigens that stimulate pre-killer cells appear functionally distinct from those heat labile antigens (Mls, I, H-2K/H-2D associated) that stimulate helper factor production since heat-treated allogeneic cells served as stimulators of cytotoxicity provided helper activity was added to the MLC.     

Circumvention of defective CD4 T helper cell function in HIV-infected individuals by stimulation with HLA alloantigens

PBL from approximately 50% of asymptomatic individuals infected with HIV have been previously demonstrated to exhibit defective in vitro Th function that is selective for influenza A virus (FLU), but not for HLA alloantigens (ALLO). In this report, we have further studied HIV+ individuals with this selective Th defect, and demonstrate that defective in vitro CTL responses to FLU can be restored by costimulation with FLU + ALLO. In contrast, HIV+ patients who have lost Th responses to ALLO were unable to correct CTL responses to FLU by this costimulation procedure. These findings indicate that intact Th responses to ALLO can be used in vitro to provide Th signals necessary to activate the T effector cell response to a potential pathogenic virus. Our results raise the possibility that a program of in vivo coimmunization with ALLO plus antigens of potential pathogens (including HIV) can be useful in HIV+ patients exhibiting selective defects in Th function. Furthermore, this approach could be incorporated in vaccine trials aimed at enhancing immunity to HIV in patients who have been infected previously with this virus.