Heterogeneity of B cell clones: immunoglobulin-secreting subsets

Staphylococcus aureus Cowan I bacteria (SpA CoI) is known to be a polyclonal B-cell activator of human lymphocytes. In this study, we investigated which of the B-cell subsets SpA CoI could stimulate and induce immunoglobulin (Ig) production. B-Cell subsets were separated from peripheral blood and tonsil lymphocytes by rosette formation with E, EA_IgG, EAC, anti-Ig-conjugated ox erythrocytes (OE-anti-Ig), and protein A-conjugated OE (OE-Pro A), or on a bovine serum albumin (BSA) discontinuous density gradient. The cells responding to SpA CoI included E^?, C3 receptor-positive (C3R⁺), Fc receptor-negative (FcR^?), and surface Ig-positive (SIg⁺) B-cell subsets. These B-cell populations responded well to SpA CoI and produced significant amounts of IgG, IgM, and a lesser amount of IgA. Among SIg⁺ B cells, IgG, IgA, and IgM⁺ B-cell subsets responded to SpA CoI and produced large amounts of Ig belonging to each corresponding Ig class. IgD⁺ B cells failed to produce Ig of any class, except for minimal amounts of IgG and IgM. While both the protein A receptor-positive (Pro A · R⁺) and negative (Pro A · R^?) cells responded well to SpA CoI, Pro A · R⁺ B cells produced IgG mainly and Pro A · R^? B cells produced IgM. Fractionation of B cells on a BSA gradient revealed that comparatively small-sized and denser B-cell subsets responded well to SpA CoI and produced every class of Ig.     

Influence of human T lymphocytes identified by antibodies to dipeptidyl peptidase IV on differentiation of human B lymphocytes stimulated with Staphylococcus aureus Cowan I and pokeweed mitogen

The influence of human T lymphocytes expressing the enzyme dipeptidyl peptidase IV (DPP IV) was investigated with respect to human peripheral B-lymphocyte differentiation. B cells stimulated with pokeweed mitogen in the presence of DPP IV-positive T cells produced high amounts of immunoglobulin. Moderate amounts of immunoglobulin could be measured when B cells were cultured in the presence of DPP IV-negative T cells. DPP IV defines a T-cell subset partially overlapping the subsets characterized by the differentiation antigens Leu 3a (helper/inducer) and Leu 2a (suppressor/cytotoxic). DPP IV-positive T cells exert, in contrast to DPP IV-negative T cells, high interleukin-2 activity after stimulation with phytohemagglutinin and pokeweed mitogen. To further functionally characterize DPP IV-positive and DPP IV-negative T cells, the helper effects of Leu 3a-positive T-cell subsets, differing in DPP IV expression, were investigated in pokeweed mitogen- and Staphylococcus aureus-driven B-cell differentiation systems. After pokeweed mitogen stimulation, immunoglobulin production was markedly reduced when B cells were cultured in the presence of Leu 3a-positive T cells expressing DPP IV (DPP IV+/Leu 3a+). In contrast, high amounts of immunoglobulin were produced in cultures with Leu 3a-positive but DPP IV-negative T cells (DPP IV-/Leu 3a+). This difference in immunoglobulin production of B cells cultured with DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells could not be observed in Staphylococcus aureus-stimulated cultures. Here, both T-cell subsets supported terminal differentiation of B cells. We conclude that in the pokeweed mitogen-driven culture systems, DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells may differ in the production of growth and/or differentiation factors distinct from interleukin-2.     

Autoregulatory function of interleukin-10-producing pre-na?ve B cells is defective in systemic lupus erythematosus

IntroductionPre-naïve B cells represent an intermediate stage in human B-cell development with some functions of mature cells, but their involvement in immune responses is unknown. The aim of this study was to determine the functional role of normal pre-naïve B cells during immune responses and possible abnormalities in systemic lupus erythematosus (SLE) that might contribute to disease pathogenesis.MethodsPre-naïve, naïve, and memory B cells from healthy individuals and SLE patients were stimulated through CD40 and were analyzed for interleukin-10 (IL-10) production and co-stimulatory molecule expression and their regulation of T-cell activation. Autoreactivity of antibodies produced by pre-naïve B cells was tested by measuring immunoglobulin M (IgM) autoantibodies in culture supernatants after differentiation.ResultsCD40-stimulated pre-naïve B cells produce larger amounts of IL-10 but did not suppress CD4⁺ T-cell cytokine production. Activated pre-naïve B cells demonstrated IL-10-mediated ineffective promotion of CD4⁺ T-cell proliferation and induction of CD4⁺FoxP3⁺ T cells and IL-10 independent impairment of co-stimulatory molecule expression and tumor necrosis factor-alpha (TNF-α) and IL-6 production. IgM antibodies produced by differentiated pre-naïve B cells were reactive to single-stranded deoxyribonucleic acid. SLE pre-naïve B cells were defective in producing IL-10, and co-stimulatory molecule expression was enhanced, resulting in promotion of robust CD4⁺ T-cell proliferation.ConclusionsThere is an inherent and IL-10-mediated mechanism that limits the capacity of normal pre-naïve B cells from participating in cellular immune response, but these cells can differentiate into autoantibody-secreting plasma cells. In SLE, defects in IL-10 secretion permit pre-naïve B cells to promote CD4⁺ T-cell activation and may thereby enhance the development of autoimmunity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0687-1) contains supplementary material, which is available to authorized users.     

Polysaccharides with sulfate groups are human T-cell mitogens and murine polyclonal B-cell activators (PBAs). I. Fucoidan and heparin

We reported previously that dextran sulfate and carrageenan (kappa, lambda, and iota), which are sulfated polysaccharides, were human T-cell mitogens and mouse polyclonal B-cell activators (PBA). To clarify our working hypothesis further, we used fucoidan and heparin, both sulfated polysaccharides. The following results were obtained: (1) fucoidan is human T-cell mitogen and a mouse PBA; (2) heparin is also a human T-cell mitogen and a mouse PBA, but the degree of the responses by heparin is lower than that by fucoidan; (3) helper T-cell-dependent B-cell differentiation was not observed, since both fucoidan and heparin activate OKT4⁺ cells and OKT8⁺ cells nonspecifically and suppressor T cells (OKT8⁺ cells) may inhibit the helper function of B-cell differentiation by helper T cells (OKT4⁺ cells); and (4) our working hypothesis that polysaccharides with sulfate groups are human T-cell mitogens and mouse PBAs was further strengthened. The relationship between molecular weight and sulfate groups of the polysaccharides is discussed in detail.     

Ia-positive T lymphocytes are the producer cells of interferon gamma

The production of γ-interferon (IFNγ) in human peripheral blood T lymphocytes was induced by stimulation with PHA. For identification of the producer cell of IFNγ, double fluorescence studies were undertaken and titers of interferon were determined in preparatively separated T-cell subpopulations reactive with one of the monoclonal antibodies OKT3, OKT4, OKT8, and OKIa1. Production of IFNγ was found in OKT3⁺, OKT4⁺, and OKT8⁺ cells. However, IFNγ production occurred only in T cells also reactive with the monoclonal antibody OKIa1. Addition of macrophages had no substantial effect on interferon titers in these subpopulations. It is suggested that the T cell subset producing IFNγ is characterized by its reactivity with the monoclonal antibodies OKT3, OKT4 or OKT8, and OKIa1.     

Functional analysis of monkey lymphocyte subsets defined by OKT4 and OKT8 monoclonal antibodies

The percentages of rhesus monkey blood lymphocytes (PBL) reactive with OKT4 and OKT8 antibodies and the $\text{OKT}\text{4}\text{OKT}\text{8}$ ratio showed significant correlations with the log of the immunoglobulin plaque-forming cell (PFC) response after stimulation with pokeweed mitogen (PWM). These correlations suggested that monkey OKT4⁺ cells function as “helper” cells and OKT8⁺ cells function as “suppressor” cells for the PFC response. This was confirmed by separation and study of enriched T- and B-cell subpopulations. OKT8-depleted (OKT4⁺) and OKT4-depleted (OKT8⁺) cells were obtained by treatment of purified T cells with antibody and complement. OKT4⁺ cells augmented the PWM-induced B-cell differentiation into PFC but OKT8⁺ cells did not. OKT8⁺ cells suppressed the PFC response by mixtures of B cells and OKT4⁺ cells. OKT8 antibodies also detected a suppressive cell subset in African green monkeys since the percentage of OKT8⁺ cells showed a negative correlation with the log PFC response. OKT4 antibodies failed to bind to African green monkey PBL.     

High levels of memory B cells are associated with response to a first tumor necrosis factor inhibitor in patients with rheumatoid arthritis in a longitudinal prospective study

Introduction

Tumor necrosis factor inhibitor (TNFi) therapy is effective for rheumatoid arthritis (RA). Some researchers have suggested that TNFi therapy affects B-cell homeostasis. We studied the effect of TNFi therapy on the distribution of peripheral B-cell subsets to elucidate B-cell–related biomarkers to predict the TNFi response.

Methods

Peripheral B cells were analyzed for expression of CD19, CD27, CD38 and immunoglobulin D in 31 healthy donors and 96 RA patients, including 21 patients who were followed 3 months after TNFi initiation.

Results

Treatment with steroids significantly altered the distribution of B-cell subsets. After we adjusted for age, sex and steroid dose, we found that patients with RA had B-cell subset proportions similar to controls. B-cell subset distributions did not differ upon use of TNFi at baseline or before or after TNFi introduction. TNFi responders (according to European League Against Rheumatism criteria) at 3 months had significantly higher proportions of CD27⁺ memory B cells at baseline, and ≥26% CD27⁺ cells at inclusion was associated with a relative risk of 4.9 (1.3 to 18.6) for response to TNFi treatment. CD27⁺ cells produced three times more TNFα than did TNFi-naïve B cells and were correlated with interferon γ produced from CD4⁺ cells in patients without TNFi treatment.

Conclusions

In patients with RA, high levels of baseline memory B cells were associated with response to TNFi, which may be related to TNFα-dependent activation of the T helper type 1 cell pathway.     

Tim-3-Expressing CD4+ and CD8+ T Cells in Human Tuberculosis (TB) Exhibit Polarized Effector Memory Phenotypes and Stronger Anti-TB Effector Functions

T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4⁺ and CD8⁺ T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3⁻ counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4⁺ and CD8⁺ T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.     

Human T-cell-mediated cytotoxicity: Role of subsets and neutralization of cytotoxicity by anti-α-lymphotoxin serum

T cells were isolated from peripheral blood lymphocytes (PBL) and sensitized to allogeneic PBL in a one-way mixed lymphocyte culture. The sensitized cells were fractionated on the basis of the presence of Fc receptors for IgG (T_G+) or IgM (T_M+), or the absence of both IgG and IgM receptors (T_G?,M?). The Cytotoxicity of the T cells was found to reside principally in the T_G?,M? subset. The degree of target cell lysis by this subset was related to the effector-to-target cell ratio. The sensitized T cells were also separated into subsets by treatment with monoclonal OKT4 antibody and complement (yielding OKT8⁺ cells) or OKT8 antibody and complement (yielding OKT4⁺ cells). The OKT8⁺ subset was the more cytotoxic of the two subsets, and this Cytotoxicity was again related to the effector-to-target cell ratio. The T-cell subsets obtained by these methods were characterized by immunologie and morphologic means. The Cytotoxicity of the total sensitized T-cell population or the T_G?,M? subset could be neutralized to a considerable extent by anti-human α-lymphotoxin (anti-α-LT) serum, and α-LT thus appears to have an important role in cytolysis in this system.     

Enterovirus-Infected β-Cells Induce Distinct Response Patterns in BDCA1+ and BDCA3+ Human Dendritic Cells

Enteroviruses often cause mild disease, yet are also linked to development of autoimmune diabetes. Dendritic cells (DCs) shape both innate and adaptive immune responses, including anti-viral responses. How different human DC subsets shape anti-viral responses, whether they have complementary or overlapping functions and how this relates to autoimmune responses is largely unknown. We used enterovirus-infected β-cells and freshly isolated human myeloid DC (mDC) subsets as a model for autoimmune type 1 diabetes. Our data show that both the BDCA1⁺ and BDCA3⁺ mDC subsets engulf mock- as well as virus-infected β-cells, albeit BDCA1⁺ mDCs are more efficient. Uptake of enterovirus-infected, but not mock-infected cells, activated both DC subsets as indicated by the induction of co-stimulatory molecules and secretion of type I and type III interferons. Both subsets produced similar amounts of interferon-α, yet the BDCA3⁺ DC were superior in IFN-λ production. The BDCA1⁺ mDCs more strongly upregulated PD-L1, and were superior in IL-12 and IL-10 production as compared to the BDCA3⁺ DC. Despite lack of IL-12 production by the BDCA3+ DC, both BDCA1⁺ and BDCA3⁺ DCs activated T cells in allogeneic mixed lymphocyte reaction towards a Th1-type reactivity while suppressing Th2-associated cytokines.     

Immunorestorative properties of thymostimulin (TS) in patients with Hodgkin's disease in clinical remission

Summary Fifteen Hodgkin's disease patients (8 male, 7 female) aged 19–72 years, who had been in complete unmaintained remission for 1 year or more when the study was initiated, were given 50 mg thymostimulin (TS) IM daily for 60 consecutive days. When compared with 26–30 age- and sex-matched controls, as a group the patients' circulating E_NR⁺, OKT ₃ ⁺ , and OKT ₄ ⁺ cells were depressed (0.001P .06), whereas their OKT ₈ ⁺ cell population was not. Low (>1 SD or >2 SD below mean in controls) or borderline (mean value of two subsequent tests >1 SD below mean in controls) values of E_NR⁺, OKT ₃ ⁺ , and OKT ₄ ⁺ cells were seen in nine (group I) of the 15 patients tested, while the remaining six patients (group II) had normal T-cell proportions. Following TS treatment, the proportions of E_NR⁺, OKT ₃ ⁺ , and OKT ₄ ⁺ cells increased to normal in all group I patients. The T-cell levels, however, decreased to pretreatment values 60–70 days after completion of TS therapy. TS had no effect on the group II patients whose T-cell percentages had initially been normal. Spontaneous cell-mediated cytotoxicity (SCMC) was assessed in 11 patients, and irrespective of the baseline values, there was a significant enhancement (P<0.005) by day 15 of TS administration, which was maintained during treatment. SCMC, however, returned to pretreatment levels 60–70 days after TS was discontinued. The delayed skin test reactivity to DNCB was significantly depressed in all cases. Although TS restored the T-cell proportions, it failed to reverse DNCB reactivity from negative to positive in any of the patients tested. TS can thus restore defective T-cell frequencies and can enhance cytolytic functions that are potentially important in host immunosurveillance, but it apparently failed to improve the skin reactivity to neoantigen.     

Role of individual chains of HLA-DR antigens in activation of T cells induced by alloantigens

The role of HLA-DR antigens in the activation of T cells in the allogeneic mixed lymphocyte reaction (MLR) was studied by using antibodies raised against the alpha, beta or the complex of both chains of the HLA-DR antigens. Antisera directed against the alpha or the beta chain strongly inhibited the T-cell proliferative response when added at the begining of MLR cultures but not 72 h later. T cells from MLR cultures treated with either alpha-chainor beta-chain-specific antibodies did not respond to interleukin-2 (IL-2) by proliferating, whereas T cells from non-anti-DR-treated cultures showed a proliferative response to IL-2 stimulation. However, neither the anti-alpha chain nor the anti-beta chain serum was able to inhibit continuous proliferation of already activated, IL-2-reactive T cells supported by IL-2. In MLR, OKT4⁺ but not OKT8⁺ lymphocytes synthesized IL-2. This function was abrogated by the alpha-chain-specific antibody but not by the anti-beta chain serum. Interleukin-1 (IL-1) did not reverse the inhibitory activity on IL-2 synthesis of the alpha-chain antibody, while IL-1 promoted the production of IL-2 in MLR cultures not exposed to the anti-DR sera. In addition, nonstimulated OKT4⁺ cells were unresponsive to IL-1 and did not produce IL-2. From these results, it is concluded that HLA-DR antigens participate actively in the activation of T cells by allogeneic non-T cells. Thus, both the alpha and beta chains of HLA-DR antigens render resting T cells sensitive to IL-2. In addition, the alpha but not the beta chain participates in the production of IL-2 by enabling OKT4⁺ lymphocytes to respond to IL-1 and subsequently to synthesize IL-2. Once T cells have acquired responsiveness to IL-2 and this growth factor has been produced there is no further requirement for HLA-DR antigens. Continuous proliferation and growth of IL-2-reactive T cells depends on the availability of interleukin-2.     

A randomized trial to evaluate the immunorestorative properties of thymostimulin in patients with Hodgkin's disease in complete remission

Summary A total of 19 Hodgkin's disease (HD) patients (12 male, 7 female) aged 26–67 years, who had been in complete unmaintained remission for 6 months or more when the study was initiated, were randomly given 50 mg thymostimulin (TS) i.m. daily (G1) or every other day (G2) for 35 days. A third group (G3) was not treated. Then TS, at the same dose was administered twice a week for the following 22 weeks in patients both initially receiving loading or intermittent TS treatment. When compared with age-and sex-matched controls, as a group, the patients' circulating OKT ₃ ⁺ , OKT ₄ ⁺ , OKT ₁₁ ⁺ and E-AETR⁺ cells were depressed (P<0.001 for both proportions and absolute numbers), whereas their OKT ₈ ⁺ cell population was not. Following 5 weeks of daily TS administration, the proportions and numbers of all T cell fractions significantly increased in G1 patients (P<0.03 for all the comparisons tested), while following intermittent TS treatment (G2) only the proportions of OKT ₃ ⁺ and OKT ₁₁ ⁺ cells (P<0.03), but not of other T cell fractions, significantly increased. In addition, no significant changes in the absolute numbers of T cell fractions were observed in this group of patients. Furthermore, no spontaneous variations in the T cell pool size occurred in untreated patients. TS maintenance therapy did not produce any further improvement in the size of overall T cells and T cell subsets but sustained percentage and absolute numbers of these cells during administration and the absolute number of T cells even after discontinuation of therapy. The TS-induced improvement in the T cell pool was not associated with any change in the size of circulating non-T lymphocytes and monocytes. In vitro phytohemagglutinin-induced interleukin-2 (IL-2) and gamma-interferon (IFN-) synthesis was assessed in 11 patients (3 G1, 4 G2, and 4 G3). Although it was not statistically significant, a rise in IL-2 and IFN- production was observed in TS-treated patients, but not in untreated controls. TS failed to exert any effect on the serum circulating levels of neopterin, type I and II IFN, beta-2 microglobulin (B₂-M) and immunoglobulins (Ig). TS can thus improve defective T cell frequences and numbers and may modulate IL-2 and IFN- production.     

Impairment of Pneumococcal Antigen Specific Isotype-Switched Igg Memory B-Cell Immunity in HIV Infected Malawian Adults

Pneumococcal disease is associated with a particularly high morbidity and mortality amongst adults in HIV endemic countries. Our previous findings implicating a B-cell defect in HIV-infected children from the same population led us to comprehensively characterize B-cell subsets in minimally symptomatic HIV-infected Malawian adults and investigate the isotype-switched IgG memory B-cell immune response to the pneumococcus. We show that similar to vertically acquired HIV-infected Malawian children, horizontally acquired HIV infection in these adults is associated with IgM memory B-cell (CD19⁺ CD27⁺ IgM⁺ IgD⁺) depletion, B-cell activation and impairment of specific IgG B-cell memory to a range of pneumococcal proteins. Our data suggest that HIV infection affects both T-cell independent and T-cell dependent B-cell maturation, potentially leading to impairment of humoral responses to extracellular pathogens such as the pneumococcus, and thus leaving this population susceptible to invasive disease.     

Human “Orchestrator” CD11b+ B1 Cells Spontaneously Secrete Interleukin-10 and Regulate T-Cell Activity

Immune regulation produced by B cells has been attributed to production and secretion of interleukin (IL)-10, which is a characteristic of mouse B1 cells. In view of the widespread clinical use of B-cell depletion therapies in autoimmune and malignant diseases, it is important to monitor the function and fate of regulatory B cells. However, there is no consensus regarding the phenotypic identity of human IL-10⁺ B cells. Here we show that human CD11b⁺ B1 cells, one of two recently described subpopulations of B1 cells, spontaneously produce IL-10 and suppress T-cell activation. In view of the capacity of these B cells to either stimulate T-cell proliferation or suppress T-cell activation, CD11b⁺ B1 cells are considered to be capable of orchestrating elements of immune responsiveness and thus are termed “orchestrator B1 cells,” or “B1orc,” whereas CD11b⁻ B1 cells that primarily secrete antibody are termed “secretor B1 cells,” or “B1sec.”     

History of immunology. Development of the concept of immunologic specificity, I

The induction of differentiation in human malignant T-lymphoblastic cell lines MOLT-3 and Jurkat by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined using the monoclonal antibodies OKT3, OKT4, OKT6, and OKT8 which are known to react with human T-cell differentiation antigens. It was found that in the presence of nanomolar concentrations of TPA the proportion of OKT3⁺ (mature T-cell marker) cells increased while the proportion of OKT4⁺, OKT6⁺, and OKT8⁺ (relatively immature T-cell markers) cells decreased. These changes in the distribution of the OKT antigens in MOLT-3 cells were found to be more prominent with MOLT-3 cells than when the Jurkat cells were used. In studies using a double labeling approach it was found that although the OKT3⁺ and E-rosette-positive (E⁺) cells appeared to belong to the same subpopulations of MOLT-3 cells, the OKT3 antigen was probably not related to the receptor for sheep erythrocytes because adsorption of the OKT3 antibody did not block E-rosette formation. Studies using the DNA synthesis inhibitor, arabinosylcytidine (ara-C) also indicate that DNA synthesis was not required for the induction of more mature T-cell antigens in the malignant T-cell lines by TPA. These studies, taken together with our earlier reports, support the conclusion that namomolar concentrations of TPA can induce differentiation in these malignant T-cell lines. Furthermore we have shown that the T-cell hybridoma antibodies are useful markers to detect differentiation changes in human T cells.     

T-cell receptor Vβ repertoire of CD8+ T-lymphocyte subpopulations in cutaneous leishmaniasis patients from the state of Rio de Janeiro, Brazil

In human cutaneous leishmaniasis (CL), the immune response is mainly mediated by T-cells. The role of CD8⁺ T-lymphocytes, which are related to healing or deleterious functions, in affecting clinical outcome is controversial. The aim of this study was to evaluate T-cell receptor diversity in late-differentiated effector (LDE) and memory CD8⁺ T-cell subsets in order to create a profile of specific clones engaged in deleterious or protective CL immune responses. Healthy subjects, patients with active disease (PAD) and clinically cured patients were enrolled in the study. Total CD8⁺ T-lymphocytes showed a disturbance in the expression of the Vβ2, Vβ9, Vβ13.2, Vβ18 and Vβ23 families. The analyses of CD8⁺T-lymphocyte subsets showed high frequencies of LDE CD8⁺T-lymphocytes expressing Vβ12 and Vβ22 in PAD, as well as effector-memory CD8⁺ T-cells expressing Vβ22. We also observed low frequencies of effector and central-memory CD8⁺ T-cells expressing Vβ2 in PAD, which correlated with a greater lesion size. Particular Vβ expansions point to CD8⁺ T-cell clones that are selected during CL immune responses, suggesting that CD8⁺ T-lymphocytes expressing Vβ12 or Vβ22 are involved in a LDE response and that Vβ2 contractions in memory CD8⁺T-cells are associated with larger lesions.     

The regulatory roles of T4 and T8 subsets in tetanus toxoid-induced in vitro immunoglobulin production

A new mitogenic system for in vitro immunoglobulin production induced by tetanus toxoid is presented and the role of T4 and T8 subsets in tetanus toxoid-induced in vitro immunoglobulin production is investigated. Purified T, T4, T8, and B cells from normal individuals previously immunized but not boosted with tetanus toxoid were cultured in helper and suppressor assays and the number of immunoglobulin-secreting cells were enumerated after culture using a hemolytic plaque assay. The regulatory roles of T4 and T8 cells in this tetanus toxoid system were compared with the role of these subsets after pokeweed mitogen stimulation. Although most of the immunoglobulin produced in the tetanus toxoid system was polyclonal, there were differences in the time course, the magnitude of the responses, the radiosensitivity of the subsets, and optimal T- to B-cell ratios for immunoglobulin production which distinguish the tetanus toxoid and pokeweed mitogen systems.     

Effects of Combined CCR5/Integrase Inhibitors-Based Regimen on Mucosal Immunity in HIV-Infected Patients Na?ve to Antiretroviral Therapy: A Pilot Randomized Trial

Whether initiation of antiretroviral therapy (ART) regimens aimed at achieving greater concentrations within gut associated lymphoid tissue (GALT) impacts the level of mucosal immune reconstitution, inflammatory markers and the viral reservoir remains unknown. We included 12 HIV- controls and 32 ART-naïve HIV patients who were randomized to efavirenz, maraviroc or maraviroc+raltegravir, each with fixed-dose tenofovir disoproxil fumarate/emtricitabine. Rectal and duodenal biopsies were obtained at baseline and at 9 months of ART. We performed a comprehensive assay of T-cell subsets by flow cytometry, T-cell density in intestinal biopsies, plasma and tissue concentrations of antiretroviral drugs by high-performance liquid chromatography/mass spectroscopy, and plasma interleukin-6 (IL-6), lipoteichoic acid (LTA), soluble CD14 (sCD14) and zonulin-1 each measured by ELISA. Total cell-associated HIV DNA was measured in PBMC and rectal and duodenal mononuclear cells. Twenty-six HIV-infected patients completed the follow-up. In the duodenum, the quadruple regimen resulted in greater CD8⁺ T-cell density decline, greater normalization of mucosal CCR5⁺CD4⁺ T-cells and increase of the naïve/memory CD8⁺ T-cell ratio, and a greater decline of sCD14 levels and duodenal HIV DNA levels (P = 0.004 and P = 0.067, respectively), with no changes in HIV RNA in plasma or tissue. Maraviroc showed the highest drug distribution to the gut tissue, and duodenal concentrations correlated well with other T-cell markers in duodenum, i.e., the CD4/CD8 ratio, %CD4⁺ and %CD8⁺ HLA-DR⁺CD38⁺ T-cells. Maraviroc use elicited greater activation of the mucosal naïve CD8⁺ T-cell subset, ameliorated the distribution of the CD8⁺ T-cell maturational subsets and induced higher improvement of zonulin-1 levels. These data suggest that combined CCR5 and integrase inhibitor based combination therapy in ART treatment naïve patients might more effectively reconstitute duodenal immunity, decrease inflammatory markers and impact on HIV persistence by cell-dependent mechanisms, and show unique effects of MVC in duodenal immunity driven by higher drug tissue penetration and possibly by class-dependent effects.