A Randomized Placebo-Controlled Efficacy Study of a Prime Boost Therapeutic Vaccination Strategy in HIV-1-Infected Individuals: VRI02 ANRS 149 LIGHT Phase II Trial.

In this placebo-controlled phase II randomized clinical trial, 103 human immunodeficiency virus type 1 (HIV-1)-infected patients under cART (combined antiretroviral treatment) were randomized 2:1 to receive either 3 doses of DNA GTU-MultiHIV B (coding for Rev, Nef, Tat, Gag, and gp160) at week 0 (W0), W4, and W12, followed by 2 doses of LIPO-5 vaccine containing long peptides from Gag, Pol, and Nef at W20 and W24, or placebo. Analytical treatment interruption (ATI) was performed between W36 to W48. At W28, vaccinees experienced an increase in functional CD4+ T-cell responses (P < 0.001 for each cytokine compared to W0) measured, predominantly against Gag and Pol/Env, and an increase in HIV-specific CD8+ T cells producing interleukin 2 (IL-2) and tumor necrosis factor alpha (TNF-α) (P = 0.001 and 0.013, respectively), predominantly against Pol/Env and Nef. However, analysis of T-cell subsets by mass cytometry in a subpopulation showed an increase in the W28/W0 ratio for memory CD8+ T cells coexpressing exhaustion and senescence markers such as PD-1/TIGIT (P = 0.004) and CD27/CD57 (P = 0.044) in vaccinees compared to the placebo group. During ATI, all patients experienced viral rebound, with the maximum observed HIV RNA level at W42 (median, 4.63 log10 copies [cp]/ml; interquartile range [IQR], 4.00 to 5.09), without any difference between arms. No patient resumed cART for CD4 cell count drop. Globally, the vaccine strategy was safe. However, a secondary HIV transmission during ATI was observed. These data show that the prime-boost combination of DNA and LIPO-5 vaccines elicited broad and polyfunctional T cells. The contrast between the quality of immune responses and the lack of potent viral control underscores the need for combined immunomodulatory strategies. (This study has been registered at ClinicalTrials.gov under registration no. NCT01492985.)IMPORTANCE In this placebo-controlled phase II randomized clinical trial, we evaluated the safety and immunogenicity of a therapeutic prime-boost vaccine strategy using a recombinant DNA vaccine (GTU-MultiHIV B clade) followed by a boost vaccination with a lipopeptide vaccine (HIV-LIPO-5) in HIV-infected patients on combined antiretroviral therapy. We show here that this prime-boost strategy is well tolerated, consistently with previous studies in HIV-1-infected individuals and healthy volunteers who received each vaccine component individually. Compared to the placebo group, vaccinees elicited strong and polyfunctional HIV-specific CD4+ and CD8+ T-cell responses. However, these immune responses presented some qualitative defects and were not able to control viremia following antiretroviral treatment interruption, as no difference in HIV viral rebound was observed in the vaccine and placebo groups. Several lessons were learned from these results, pointing out the urgent need to combine vaccine strategies with other immune-based interventions.

CO2 laser and the genitourinary syndrome of menopause: a randomized sham-controlled trial.

PURPOSE: This study aimed to clarify the efficacy of intravaginal CO2-laser treatment in postmenopausal women with genitourinary syndrome of menopause (GSM). MATERIALS AND METHODS: This double-blind, randomized, sham-controlled trial included postmenopausal women diagnosed with GSM and bothersome dryness and dyspareunia. Treatment consisted of three sessions. Active CO2-laser treatments (active group) were compared to sham treatments (sham group) with the primary endpoints being changes in dryness and dyspareunia intensity, as assessed by the 10-cm visual analog scale. Secondary endpoints were as follows: changes in Female Sexual Function Index (FSFI; total score and all domains), itching, burning, dysuria, and Urogenital Distress Inventory (UDI-6); incidence of symptoms; and presence of adverse events. All outcomes were evaluated at baseline and 4 months post baseline. RESULTS: Fifty-eight women (28 in the active group and 30 in the sham group) were eligible for inclusion. In the active group, dryness, dyspareunia, FSFI (total score), itching, burning, dysuria, and UDI-6 were significantly improved (mean [standard deviation] -5.6 [2.8], -6 [2.6], 12.3 [8.9], -2.9 [2.8], -2.3 [2.8], -0.9 [2.1], and -8.0 [15.3], respectively). In the sham group, dryness, itching, and burning were significantly improved (-1.9 [2], -1.4 [1.9], and -1 [1.9], respectively). All changes were in favor of the active group. After completion of the protocol, the proportion of participants with dryness, dyspareunia, and sexual dysfunction was significantly lower in the active group compared to those in the sham group (all p < 0.005). CONCLUSIONS: CO2 laser could be proposed as an effective alternative treatment for the management of GSM as it is superior to sham treatments.

Complement activation and blockade in massive post-partum haemorrhage, thrombotic microangiopathy and acute kidney injury: a case report.

BACKGROUND: Thrombotic microangiopathy (TMA)-mediated acute kidney injury (AKI) following massive haemorrhage is a rare but severe complication of the post-partum period. It is associated with a poor renal prognosis and a high risk of end-stage kidney disease. Complement activation may occur in this picture. However, whether complement activation, and thus complement blockade, may be critically relevant in this setting is unknown. CASE PRESENTATION: A 50 year-old woman presented with massive delayed post-partum haemorrhage (PPH). Despite bleeding control and normalization of coagulation parameters, she rapidly developed AKI stage 3 associated with dysmorphic microhematuria and proteinuria up to 2 g/day with the need of renal replacement therapy. Blood tests showed signs of TMA associated with markedly increased sC5b-9 and factor Bb plasma levels, respectively markers of terminal and alternative complement pathway over-activation. This clinical picture prompted us to initiate anti-C5 therapy. sC5b-9 normalized within 12 h after the first dose of eculizumab, factor Bb and C3 after seven days, platelet count after nine days and haptoglobin after 3 weeks. The clinical picture improved rapidly with blood pressure control within 48 h. Diuresis resumed after three days, kidney function rapidly improved and haemodialysis could be discontinued after the sixth and last dose. Serum creatinine returned to normal two years after presentation. CONCLUSIONS: We suggest that massive PPH induced major activation of complement pathways, which ultimately lead to TMA-induced AKI. Various causes, such as oocyte-donation, the potential retention of placental material and the use of tranexamic acid may have contributed to complement activation due to PPH. The prompt administration of anti-C5 therapy may have rapidly restored kidney microcirculation patency, thus reversing signs of TMA and AKI. We propose that complement activation may represent a major pathophysiological player of this complication and may provide a novel therapeutic avenue to improve renal prognosis in TMA-induced AKI following massive PPH.

Dental implants survival rate in controlled type I diabetic patients: a prospective longitudinal study with a 2-year follow-up.

The aim of the study was to evaluate implant treatment for partial edentulism in a population of controlled type I diabetic patients. The research hypothesis was that implant survival rate, prevalence of peri-implant tissue infection and marginal bone loss at 2 years follow-up would not differ from a non diabetic population. A total of 106 patients (47=women, 59=men, mean age 38.36 years) presented with partially edentulous jaws. All patients underwent a two stage implant surgery (105 maxillary, 100 mandibular). Diabetic type I patients (53) were scheduled in Group A, while 53 healthy patients formed the Control Group. Clinical and radiological controls were performed from baseline up to 24 months and implants survival rate, presence of peri-implant tissue infections and marginal bone loss were assessed in all ptients. Group A and Control Group were compared by analyzing data at implant level, through either an independent sample t-test, with respect to bone loss, or Fisher Exact tests, with respect to (a) peri-implant mucositis, (b) peri-implantitis, and (c) post-operative wound infection. At the 24-month follow-up, 5 and 3 implants failed in diabetic and non-diabetic patients, respectively. No statistically significant difference was found in implant survival rate between the two groups (Group A: 95.19%; Control Group: 97.03%). Moreover, no statistical significant differences were found in infections occurrence, nor in marginal bone loss. The preliminary results of this prospective study showed how implant treatment for partial edentulism may a be safe and predictable procedure for diabetic type I patients, provided controlled glycemic levels and regular professional oral hygiene sessions.

The association between minor recurrent aphthous stomatitis (RAS), children's poor oral condition, and underlying negative psychosocial habits and attitudes towards oral hygiene.

BACKGROUND: Minor Recurrent Aphthous Stomatitis (RAS) represents a disease which is very difficult to prevent. This case-control study focused on possible associations between minor Recurrent Aphthous Stomatitis in children, their oral health, and underlying behavioral indexes of children's attitudes and habits pertaining to (home) oral hygiene, with the further goal of enabling the dentist to prevent these specific kind of lesions, both from a clinical and a broader psychosocial perspective. METHODS: Four hundred one school-children (5-10 years old) in Milan (Italy) were submitted to an intra-oral examination, and interviewed with the aid of a brief psychosocial questionnaire. RESULTS: At the clinical level, statistically significant associations were observed between the presence of decayed teeth and minor Recurrent Aphthous Stomatitis (Odds Ratio: 3.15; 95% CI: lower limit 1.06; upper limit: 9.36; Z-test: 2.07, p = 0.039; Chi-square = 4.71, p = 0.030), and between the Decayed Missing or Filled Teeth (DMFT) index and minor aphthous stomatitis (Odds Ratio: 3.30; 95% CI: lower limit 1.13; upper limit: 9.67; Z-test = 2.18, p = 0.029; Chi-square = 5.27; p = 0.022), both results pointing to a significant increase-by circa 3 times-in the risk of developing minor Recurrent Aphthous Stomatitis in children exposed to the two above-identified factors (i.e., the presence of decayed teeth and a clearly compromised oral condition, as signaled by the DMFT index), if compared with the risk run by their non-exposed counterparts. At the psychosocial level of analysis, statistically significant associations were observed (1) between children's practice of spontaneously brushing teeth when not at home and a comparatively lower (i.e. better) Decayed Missing or Filled Teeth index (Chi-square: 8.95; p = 0.011), and (2) between receiving parental aid (e.g., proper brushing instructions) while practicing home oral hygiene and a significantly reduced presence of decayed teeth (Chi-square = 5.40; p = .067; Spearman's Rho, p = .038). Further, significant associations were also observed between children's reported severity of dental pain and both (a) the presence of decayed teeth (Chi-square = 10.80; p = 0.011), and (b) children's (poor) oral health condition as expressed by the Decayed Missing or Filled Teeth index (Chi-square = 6.29; p = 0.043). Interestingly, specific lifestyles and social status, showed no systematic association to other clinical or psychological/psychosocial indices. CONCLUSIONS: These systematic relations suggest that, in the presence of Recurrent Aphthous Stomatitis in pediatric patients, the dentist should carefully monitor children for potential carious lesions, implement protocols of prevention to control Recurrent Aphthous Stomatitis disease in children affected by caries, and also be particularly aware of the right or wrong habits children may acquire in the course of continued social exchange with their caregivers and peers.

The association between three attitude-related indexes of oral hygiene and secondary implant failures: A retrospective longitudinal study.

OBJECTIVE: This study evaluated the strength of the association between three widely used clinical indexes considered as distal behavioural indicators of attitude-related oral status (an index of oral hygiene, the plaque index [PI] and two periodontal indexes, that is the presence of bleeding on probing [BOP] and of pockets probing depth [PPD]) and secondary implant failure due to peri-implantitis in patients rehabilitated with cemented prosthesis. MATERIALS AND METHODS: The study included patients who underwent implant-prosthetic rehabilitation and had joined the programme of maintenance of the same hospital. Implant failures, number of months between implant insertion and implant loading, and patients' surgical protocol were monitored and recorded. Further, PI, BOP and PPD-all attitude-related indicators of oral hygiene and periodontal inflammation-were recorded and related, in terms of odds ratios (ORs ) and corresponding risk factors, to secondary implant failures. RESULTS: A total of 1427 patients (2673 implants) were enrolled. The follow-up ranged from 1.5 to 9 years (mean 5.3 years±1.3). The cumulative survival rate was 98.01%. Thirty-two patients (36 implants, 1.36% of all implants) had implant failure. A statistically significant association between PI, BOP, PPD and secondary failures due to peri-implantitis was observed. CONCLUSION: Within the limitations of this study, all three attitude-related behavioural indicators-the plaque index (PI), bleeding on probing (BOP) and abnormal probing pocket depth (PPD)-proved to be significant risk indicators for secondary implant failure due to peri-implantitis, both from a clinical and from a socio-psychological attitude-related perspective.

Performance evaluation of a new fourth-generation HIV combination antigen-antibody assay.

Education and diagnostic tests capable of early detection represent our most effective means of preventing transmission of human immunodeficiency virus (HIV). The importance of early detection is underlined by studies demonstrating increased life expectancy following early initiation of antiviral treatment. The Elecsys(®) HIV combi PT assay is a fourth-generation antigen-antibody combination assay developed to allow earlier detection of seroconversion, and to have increased sensitivity and improved specificity. We aimed to determine how early the assay could detect infection compared with existing assays; whether all HIV variants could be detected; and the assay's specificity using samples from blood donors, routine specimens, and patients with potential cross-reacting factors. Samples were identified as positive by the Elecsys(®) assay 4.9 days after a positive polymerase chain reaction result (as determined by the panel supplier), which was earlier than the 5.3-7.1 days observed with comparators. The analytical sensitivity of the Elecsys(®) HIV combi PT assay for the HIV-1 p24 antigen was 1.05 IU/mL, which compares favorably with the comparator assays. In addition, the Elecsys(®) assay identified all screened HIV subtypes and displayed greater sensitivity to HIV-2 homologous antigen and antibodies to HIV-1 E and O and HIV-2 than the other assays. Overall, the specificity of the Elecsys(®) assay was 99.88 % using samples from blood donors and 99.81 % when analyzing unselected samples. Potential cross-reacting factors did not interfere with assay performance. The Elecsys(®) HIV combi PT assay is a sensitive and specific assay that has been granted the CE mark according to Directive 2009/886/EC.

Adjunctive effect of chlorhexidine antiseptics in mechanical periodontal treatment: first results of a preliminary case series.

OBJECTIVES: The aim of the present case series was to evaluate the clinical and microbiological effects of a single session of mechanical and manual scaling and root planing (SRP) combined with the use of two different chlorhexidine formulations in the treatment for generalized chronic periodontitis. METHODS: Ten patients affected by chronic periodontal disease with periodontal probing depth (PPD) ≥ 5 mm were treated with SRP plus local chlorhexidine. In each patient, similar teeth, treated with SRP with the adjunctive use of chlorhexidine digluconate and dihydrochloride or chlorhexidine gluconate, respectively, were selected and assigned to a test and a control group. In both groups, PPD, bleeding on probing (BOP) parameters, total bacterial counts (TBC) and quality of periodontal bacteria at time 0 and 6 weeks after treatment were measured. RESULTS: PPD significantly decreased over time both in the test and in the control group; however, no significant differences between the two groups were observed. BOP and TBC were significantly lower in the test than in the control group 6 weeks after treatment. In the post-treatment revaluation, a significant decrease both in the treatment and in the control group, for each of the single periodontal pathogens, was observed. CONCLUSION: In this study--a preliminary case series with small sample size and short follow-up--the adjunctive use of chlorhexidine (CHX) to SRP resulted in clinical and microbiological benefits in the treatment for generalized chronic periodontitis. A CHX gel formulation consisting of CHX digluconate and CHX dihydrochloride seems to lead some additional benefits over SRP plus CHX gluconate in the short term. Additional investigations are needed to evaluate the effectiveness of this antiseptic therapy.

Positive and negative regulation of cellular immune responses in physiologic conditions and diseases.

The immune system has evolved to allow robust responses against pathogens while avoiding autoimmunity. This is notably enabled by stimulatory and inhibitory signals which contribute to the regulation of immune responses. In the presence of a pathogen, a specific and effective immune response must be induced and this leads to antigen-specific T-cell proliferation, cytokines production, and induction of T-cell differentiation toward an effector phenotype. After clearance or control of the pathogen, the effector immune response must be terminated in order to avoid tissue damage and chronic inflammation and this process involves coinhibitory molecules. When the immune system fails to eliminate or control the pathogen, continuous stimulation of T cells prevents the full contraction and leads to the functional exhaustion of effector T cells. Several evidences both in vitro and in vivo suggest that this anergic state can be reverted by blocking the interactions between coinhibitory molecules and their ligands. The potential to revert exhausted or inactivated T-cell responses following selective blocking of their function made these markers interesting targets for therapeutic interventions in patients with persistent viral infections or cancer.

High specificity of line-immunoassay based algorithms for recent HIV-1 infection independent of viral subtype and stage of disease.

BACKGROUND: Serologic testing algorithms for recent HIV seroconversion (STARHS) provide important information for HIV surveillance. We have shown that a patient's antibody reaction in a confirmatory line immunoassay (INNO-LIA HIV I/II Score, Innogenetics) provides information on the duration of infection. Here, we sought to further investigate the diagnostic specificity of various Inno-Lia algorithms and to identify factors affecting it. METHODS: Plasma samples of 714 selected patients of the Swiss HIV Cohort Study infected for longer than 12 months and representing all viral clades and stages of chronic HIV-1 infection were tested blindly by Inno-Lia and classified as either incident (up to 12 m) or older infection by 24 different algorithms. Of the total, 524 patients received HAART, 308 had HIV-1 RNA below 50 copies/mL, and 620 were infected by a HIV-1 non-B clade. Using logistic regression analysis we evaluated factors that might affect the specificity of these algorithms. RESULTS: HIV-1 RNA < 50 copies/mL was associated with significantly lower reactivity to all five HIV-1 antigens of the Inno-Lia and impaired specificity of most algorithms. Among 412 patients either untreated or with HIV-1 RNA ≥ 50 copies/mL despite HAART, the median specificity of the algorithms was 96.5% (range 92.0-100%). The only factor that significantly promoted false-incident results in this group was age, with false-incident results increasing by a few percent per additional year. HIV-1 clade, HIV-1 RNA, CD4 percentage, sex, disease stage, and testing modalities exhibited no significance. Results were similar among 190 untreated patients. CONCLUSIONS: The specificity of most Inno-Lia algorithms was high and not affected by HIV-1 variability, advanced disease and other factors promoting false-recent results in other STARHS. Specificity should be good in any group of untreated HIV-1 patients.

Effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo.

Mycophenolic acid, a selective inhibitor of the de novo synthesis of guanosine nucleotides in T and B lymphocytes, has been proposed to inhibit human immunodeficiency virus (HIV) replication in vitro by depleting the substrate (guanosine nucleotides) for reverse transcriptase. Here we show that mycophenolic acid induced apoptosis and cell death in a large proportion of activated CD4+ T cells, thus indicating that it may inhibit HIV infection in vitro by both virological mechanisms and immunological mechanisms (depletion of the pool of activated CD4+ T lymphocytes). Administration of mycophenolate mophetil, the ester derivate of mycophenolic acid, to HIV-infected subjects treated with anti-retroviral therapy and with undetectable viremia resulted in the reduction of the number of dividing CD4 + and CD8+ T cells and in the inhibition of virus isolation from purified CD4+ T-cell populations. Based on these results, the potential use of mycophenolate mophetil in the treatment of HIV infection deserves further investigation in controlled clinical trials.

Transfer of HIV-1-specific cytotoxic T lymphocytes to an AIDS patient leads to selection for mutant HIV variants and subsequent disease progression.

An HIV-1-seropositive volunteer was infused with an expanded autologous cytotoxic T lymphocyte (CTL) clone directed against the HIV-1 nef protein. This clone was adoptively transferred to determine whether supplementing CTL activity could reduce viral load or improve clinical course. Unexpectedly, infusion was followed by a decline in circulating CD4+ T cells and a rise in viral load. Some of the HIV isolates obtained from the plasma or CD4+ cells of the patient were lacking the nef epitope. These results suggest that active CTL selection of viral variants could contribute to the pathogenesis of AIDS and that clinical progression can occur despite high levels of circulating HIV-1-specific CTLs.

Evolutionary pattern of human immunodeficiency virus (HIV) replication and distribution in lymph nodes following primary infection: implications for antiviral therapy.

Evolutionary patterns of virus replication and distribution in lymphoid tissue during the early phases of HIV infection have not been delineated. Lymph node (LN) biopsies were excised from patients at different times after the estimated time of primary infection. Within 3 months of the acute viral syndrome, HIV was mostly present in individual virus-expressing cells in LNs; trapping of virions in the follicular dendritic cell (FDC) network was minimal or absent, but was the predominant form of HIV detected in LNs of subjects with chronic infection, either recent (4-20 months after primary infection) or long-term (>2-3 years after primary infection). Plasma viremia was significantly higher in patients during the first 3 months than in those recently infected; however, there were no significant differences in the number of virus-expressing cells per square millimeter of LN tissue in these two groups. Numbers of virus-expressing cells in lymphoid tissue were significantly lower in the subjects with long-term infection than in the other two groups. Therefore, during the transition from primary to chronic HIV infection, the level of HIV replication in lymphoid tissue remains elevated despite the fact that viremia is significantly downregulated. These findings have implications for therapeutic strategies in primary HIV infection and in recent seroconvertors.

Limited CD4+ T-cell renewal in early HIV-1 infection: effect of highly active antiretroviral therapy.

We show that the fraction of proliferating CD4+ lymphocytes is similar in HIV-infected subjects in the early stage of disease and in HIV-negative subjects, whereas the fraction of proliferating CD8+ lymphocytes is increased 6.8-fold in HIV-infected subjects. After initiation of antiviral therapy, there is a late increase in proliferating CD4+ T cells associated with the restoration of CD4+ T-cell counts. These results provide strong support for the idea of limited CD4+ T-cell renewal in the early stage of HIV infection and indicate that after effective suppression of virus replication, the mechanisms of CD4+ T-cell production are still functional in early HIV infection.

Involvement of T44 molecules in an antigen-independent pathway of T cell activation. Analysis of the correlations to the T cell antigen-receptor complex.

Prior studies indicate that the 9.3 monoclonal antibody (mAb) which defines a 44 kD T lineage-specific glycoprotein (T44) enhances the proliferative response of peripheral blood T lymphocytes to phytohemagglutinin (PHA) or allogeneic cells. The T44 molecule was expressed in both resting and activated T lymphocytes and in a subset of thymocytes, as assessed by indirect immunofluorescence and flow cytofluorometry. In view of the potential importance of T44 in T cell activation, we investigated the ability of the 9.3 (anti-T44) antibody to stimulate peripheral blood T lymphocytes under culture conditions giving optimal proliferative responses to anti-T3 mAb. Like UCHT1 (anti-T3) mAb, the 9.3 (anti-T44 mAb) promoted strong proliferative responses of purified T cells, provided that adherent cells were added to the culture. Maximal proliferation in response to 9.3 antibody was consistently detected at day 5 (at day 3 with anti-T3 or PHA). Moreover, triggering of T lymphocytes with 9.3 antibody (in the presence of adherent cells) resulted in strong IL-2 production that peaked at 48 h. Analysis of the physical and functional relationship between the T44 molecule and other molecules involved in T cell activation, including the clonotypically restricted Ti and the monomorphic T3 or T11 molecules, was carried out on a mutagenized jurkat T leukemia cell line. This mutant, termed JA3 (surface phenotype: T11+, T3+, 3A1+, T4-, T8-, DR-, Tac-, 4F2+, T44+) produced large amounts of IL-2 upon stimulation with PHA, anti-T3, or anticlonotypic mAb in conjunction with phorbol myristate acetate (or adherent cells). The molecules precipitated by anti-T44 mAb from 125I-labeled JA3 cells appeared as a diffuse band of Mr 40-45,000 under reducing conditions; under nonreducing conditions, a prominent band of Mr 80-85,000 was observed, while the Mr 40-45,000 band was greatly reduced. Thus, T44 molecules in both reducing and nonreducing conditions had relative molecular weights similar to that of molecules carrying clonotypic (Ti) determinants. In addition, like anti-Ti or anti-T3 mAb, anti-T44 antibody induced JA3 cells to produce large amounts of IL-2 in the presence of phorbol myristate acetate. Other similarities between T44 and molecules carrying clonotypic structures included the susceptibility to antibody-induced modulation and the late reexpression (72 h) at the cell surface after modulation. Taken together, these experiments suggest that anti-T44 mAb might recognize a monomorphic determinant of the T cell receptor molecule or be physically or functionally linked to the T3-Ti complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibody-induced modulation of the CD3/T cell receptor complex causes T cell refractoriness by inhibiting the early metabolic steps involved in T cell activation.

We investigated the mechanism involved in T cell unresponsiveness that follows the monoclonal antibody-induced surface modulation of the CD3-TCR complex. We determined whether modulation of CD3-TCR affected the early metabolic steps such as [Ca2+]i rise and InsP3 formation. A strong inhibition of the increase on [Ca2+]i mediated by either anti-TCR or anti-CD2 mAbs was detected. In contrast, surface modulation of CD2 molecules did not prevent the [Ca2+]i increase induced by anti-TCR mAb. Similarly, InsP3 increase was strongly reduced only after modulation of CD3-TCR complex (but not of CD2 molecules). Therefore, it appears that surface modulation of CD3-TCR complex causes T cell refractoriness by inhibiting the very early metabolic events that follow receptor-ligand interactions.

Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity.

In an attempt to determine the clonogenic properties of human peripheral blood T cells, we have developed a limiting dilution microculture system using phytohemagglutinin (PHA) as T cell activator and supernatant from PHA-stimulated spleen cultures as a source of T cell growth factors. The frequencies of cells capable of extensive proliferation under these culture conditions were 0.52-0.73, 0.98-1.11, and less than 0.02 in peripheral blood mononuclear, E-rosette-positive, and E-rosette-negative cell populations, respectively. The clonogenic potential of virtually all T cells was confirmed in experiments using single cells isolated by micromanipulation. Clone size ranged between 5 and 30 X 10(4) cells on day 14 of culture. The same microculture system was used to determine the precursor frequency of all cytolytic T lymphocytes (CTL-P). As assessed by a lectin-dependent 51Cr release assay, the CTL-P frequency in purified T cell populations ranged between 0.30 and 0.34. In comparison, the precursor frequency of T cells capable of lysing K562 target cells was ranging between 0.14 and 0.16. Parallel analysis of individual clonal cultures for both lytic activities showed that 50% of the clones exhibiting lectin-dependent lysis were also active against K562 target cells. All of the proliferating clones expressed HLA-DR antigens, although to a varying degree as assessed by flow cytofluorometry. Given the high cloning efficiency of this culture system, it appears now possible to determine the precursor frequencies of the various classes of functional cells in T cell populations.

Quantitative assessment of the pool size and subset distribution of cytolytic T lymphocytes within human resting or alloactivated peripheral blood T cell populations.

In order to directly assess the distribution of cytolytic T lymphocytes (CTL) and their precursors (CTL-P) in the two major subsets of human T cells, we have used limiting dilution microculture systems to determine their frequencies. The two subsets were defined according to their reactivity (or lack thereof) with B9.4 monoclonal antibody (the specificity of which is similar, if not identical, to that of Leu 2b monoclonal antibody). Both B9+ and B9- cells obtained by sorting peripheral blood resting T cells using the fluorescence-activated cell sorter (FACS) were assayed for total CTL-P frequencies in a microculture system that allows clonal growth of every T cell. As assessed by a lectin-dependent assay, approximately 30% of peripheral blood T cells were CTP-P. In the B9+ subset (which represents 20-30% of all T cells), the CTL-P frequency was close to 100%, whereas the B9- subset had a 25-fold lower CTL-P frequency. It is thus evident that 90% and 10% of the total CTL-P in peripheral blood are confined to the B9+ or B9- T cell subsets, respectively. Analysis of the subset distribution of CTL-P directed against a given set of alloantigens confirmed these findings. CTL-P frequencies were also determined in B9+ and B9- subsets derived from T cells that had been activated in allogenic mixed leucocyte cultures (MLC). Approximately 10% of MLC T cells were CTL-P. This frequency was increased 3.5-fold in the B9+ subset, whereas the B9- subset contained only a small, although detectable number of CTL-P. Moreover, the great majority of the (operationally defined) CTL-P in MLC T cell population were found to be directed against the stimulating alloantigens, thus indicating a dramatic increase in specific CTL-P frequencies following in vitro stimulation in bulk cultures.

Clonal heterogeneity in the requirement for T3, T4, and T8 molecules in human cytolytic T lymphocyte function.

In an attempt to define the requirement of T8, T4, and T3 surface molecules in functional interactions occurring between human cytolytic T lymphocytes (CTL) and specific target cells, we have analyzed a large number of CTL clones derived from primary mixed lymphocyte culture (MLC) T cell populations for their susceptibility to inhibition by monoclonal antibodies (mAb) directed against these surface antigens. In most experiments, MLC T cells were stained with B9.4 (anti-T8) or OKT4 (anti-T4) mAb, separated into positive and negative cells using a fluorescence-activated cell sorter (FACS) and cloned under limiting conditions. While the lytic activity of the majority of T8+ CTL clones was inhibited by B9.4 mAb, approximately 15% of these clones were unaffected even in the presence of excess antibody. Flow cytofluorometric analysis of T8 antigen in individual clones did not show any correlation between the amount of T8 antigen expressed, the magnitude of cytolytic activity and the susceptibility (or lack thereof) to inhibition by B9.4 mAb. Of the 16 T4+ CTL clones analyzed, 7 were resistant to inhibition by OKT4 mAb even at doses 10-fold higher than that sufficient for complete inhibition of susceptible clones. Again, no correlation was found between the amount of T4 antigen expressed and the susceptibility to inhibition by the corresponding antibody. The same sets of T8+ and T4+ CTL clones were also analyzed for their susceptibility to inhibition by OKT3 mAb. Although all of the clones expressed the T3 surface antigen, only 15/23 T8+ clones and 9/14 T4+ clones were inhibited by anti-T3 mAb. To further document this clonal heterogeneity, we selected two T3+ T4- T8+ CTL clones that had no concomitant NK-like activity. One clone was resistant to inhibition by OKT3 mAb, whereas the other was highly susceptible. Incubation with OKT3 mAb resulted in modulation of the T3 molecules in both clones. Following modulation, however, the cytolytic activity of the resistant clones was unaffected, whereas the lytic activity of the susceptible clone was abrogated. These results thus indicate extensive clonal heterogeneity in the requirement for T3, T4, and T8 molecules in CTL function. Moreover, it appears that T3 molecules are not always physically and functionally linked to CTL receptor structures.

Anticlonotypic monoclonal antibodies induce proliferation of clonotype-positive T cells in peripheral blood human T lymphocytes. Evidence for a phenotypic (T4/T8) heterogeneity of the clonotype-positive proliferating cells.

Three previously selected monoclonal antibodies (mAb) directed against the clonotypic structure of a variant (termed JA3) of the interleukin 2 (IL-2)-producing Jurkat leukemia cell line (anti-JTi1-3 mAb) were found to induce an adherent cell-dependent proliferation of peripheral blood T cells in 20 different donors. Unlike the early cell proliferation induced by anti-T3 mAb, anti-JTi mAb-induced proliferation was detectable at day 5-6 of culture and reached peak levels at day 7-9. Less than 1% JTi+ cells were consistently detected in the starting peripheral blood lymphocytes or in control cultures in which cells were stimulated with anti-T3, phytohemagglutinin, or allogeneic cells. However, JTi+ cells were found in increasing proportions after culture with anti-JTi mAb and they were mostly represented by large blast cells expressing either the T4 or the T8 antigen, together with typical activation antigens including HLA-DR, IL-2 receptor, and 4F2. Immunoprecipitation experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that anti-JTi-reactive molecules present on antibody-stimulated lymphocytes or on JA3 cells were similar, disulphide-linked heterodimeric structures.