Selective alterations in immunoregulatory lymphocyte subsets in early HIV (human T-lymphotropic virus type III/lymphadenopathy-associated virus) infection

In order to characterize the effects of HIV (human T-lymphotropic virus type III/lymphadenopathy-associated virus) on the immune system, Leu8^– and Leu8⁺ subsets of CD4 and CD8 cells were studied in seropositive homosexually active men without acquired immune deficiency syndrome (AIDS). Controls included both heterosexual men and HIV-seronegative homosexually active men. The decrease in CD4 levels, observed in HIV-seropositive men who were asymptomatic, as well as in those who had persistent generalized lymphadenopathy or constitutional symptoms of HIV infection, occurred proportionally in both the Leu8^– and the Leu8⁺ CD4 subsets. This observation, that HIV infection does not selectively diminish either subset of CD4 cells, indicates that the selective loss of T cell-mediated functions which accompanies the development of AIDS is not related to preferential loss of the Leu8⁺ CD4 subset. Among CD8 cells, however, HIV infection resulted in a threefold elevation in the number of Leu8^– CD8 cells, while the number of Leu8⁺ CD8 cells remained constant. The increase in Leu8^– CD8 cells was present in recent seroconverters, persistently seropositive men, and patients with AIDS. We propose that the increase in Leu8^– CD8 cells represents an HIV-specific cytotoxic T-cell response. These cells may operate by killing infected CD4 cells, thereby partially controlling viral infection while simultaneously contributing to the destruction of the immune system.     

Alterations in cytotoxic and phenotypic subsets of natural killer cells in acquired immune deficiency syndrome (AIDS)

Testing of cytotoxic function using a panel of natural killer (NK)-sensitive target cells, including a unique herpes simplex virus-infected Raji-cell target, was performed in conjunction with phenotypic cell analysis by dual-color flow cytometry to characterize the NK system. Subjects included in the study were at risk for or infected with the etiologic agent of the acquired immune deficiency syndrome (AIDS), human immunodeficiency virus (HIV). A generalized defect in NK function was temporally correlated with disease manifestations, as evidenced by deficient NK lytic function in patients with AIDS and AIDS-related complex (ARC). Healthy at-risk subjects, including those seropositive for HIV, exhibited robust NK-cell function. Phenotypic analysis revealed that normal proportions of the NK-associated CD16⁺ (Leu11) Leu7^– and CD16⁺(Leu11)Leu7⁺ lymphocyte subsets were maintained throughout the clinical progression of HIV infection. However, the proportion and numbers of cells of the CD8⁺(Leu2)Leu7⁺ subset were increased in AIDS, ARC, and healthy at-risk subjects, including those seronegative for HIV. These results are consistent with a qualitative defect in the NK system in AIDS, perhaps secondary to CD4-cell depletion and a concomitant lack of essential accessory factors. The elevation in CD8⁺(Leu2)/Leu7⁺ cells is not solely the result of HIV infection and may be a general response to viruses and/or other antigenic stimulation.     

Immunoglobulin G (IgG) Subclass Distribution and IgG1 Avidity of Antibodies in Human Immunodeficiency Virus-Infected Individuals after Revaccination with Tetanus Toxoid

In human immunodeficiency virus (HIV)-infected individuals the amount of antibodies formed after vaccination with T-cell-dependent recall antigens such as tetanus toxoid is proportional to the peripheral blood CD4⁺ T-lymphocyte counts. To investigate whether the immunoglobulin G (IgG) subclass distribution and avidity of the antibodies produced after vaccination are affected as well, we gave 13 HIV-infected adults with low CD4⁺ T-lymphocyte counts (<200 × 10⁶/liter; group I), 11 HIV-infected adults with intermediate CD4⁺ T-lymphocyte counts (≥200 × 10⁶/liter; group II), and 5 healthy controls booster immunizations with tetanus toxoid. The prevaccination antibody concentrations against tetanus toxoid were similar in the HIV-infected and healthy adults. After vaccination the total IgG and the IgG1 anti-tetanus toxoid antibody concentrations were significantly lower in group I than in group II and the controls. The avidity of the IgG1 anti-tetanus toxoid antibodies formed by HIV-infected adults was within the range for healthy controls, irrespective of their CD4⁺ T-lymphocyte counts.In human immunodeficiency virus (HIV)-infected individuals the amount of antibodies formed after vaccination with T-cell-dependent recall antigens, such as tetanus toxoid, is impaired in proportion to the number of CD4⁺ T cells and to the in vitro proliferative response of T lymphocytes to anti-CD3 monoclonal antibodies (7, 8). Protection against tetanus will depend on the total amount of antibodies, the subclass distribution, and the avidities of the antibodies that are formed. Avidity reflects the combined functional affinities of antibodies formed during a polyclonal humoral immune response and is considered to be a parameter for the efficacy of the antibodies at eliminating or neutralizing the antigen (12). The aim of the present study was to investigate whether, in addition to the concentration of antibodies, the subclass distribution and the avidity of the antibodies formed by HIV-infected individuals after booster vaccination are affected.(This study was presented in part at the 35th Annual Meeting of the Infectious Diseases Society of America, San Francisco, Calif., 13 to 16 September 1997.)     

Human Immunodeficiency Virus Type 1 Replication,Immune Activation, and Circulating CytotoxicT Cells Against Uninfected CD4+ T Cells

Cytotoxic T lymphocytes (CTL) that kill uninfected activated CD4⁺ T cells can be induced in vitro by stimulating CD8⁺ T cells with activated autologous CD4⁺ T cells. Similar CTL have been detected in circulating T cells from human immunodeficiency virus type 1 (HIV)-infected individuals. To define the in vivo correlates of this CTL activity, we studied plasma -2 microglobulin and HIV RNA levels, T-lymphocyte subset counts, and expression of CD28 on CD8⁺ T cells concurrently with circulating CTL activity against uninfected CD4⁺ T cells in 75 HIV-infected individuals at different stages of disease progression. Mean values of each parameter were compared in subsets of this group of 75 segregated on the basis of this CTL activity. The group with CTL against uninfected activated CD4⁺ T lymphocytes had more CD8⁺ T cells, a higher percentage of CD28^– CD8⁺ T cells, and higher plasma levels of HIV RNA and -2 microglobulin. CTL against uninfected activated CD4⁺ T cells were predominantly CD28^– and in HIV-infected individuals were associated with immunological or virological evidence of progressive disease. In HIV infection, circulating CTL activity against uninfected activated CD4⁺ T lymphocytes is associated with immune activation, CD8⁺ T cell expansion, accumulation of CD28^– CD8⁺ T cells, and inadequate suppression of HIV replication.     

Lysis of CD4+ lymphocytes by non-HLA-restricted cytotoxic T lymphoe from HIV-infected individuals

Individuals infected with HIV have elevated numbers of total and activated CD8⁺ lymphocytes in peripheral blood. CD8⁺ lymphocytes from HIV-infected individuals have been shown to mediate non-human histocompatibility-linked antigen (HLA)-restricted suppression of viral replication, HLA-restricted killing of cells expressing HIV antigens, and killing of uninfected lymphocytes. We studied CD8⁽ T lymphocytes that lysed autologous CD4+ lymphocytes, hetcrologous CD4¹ lymphocytes from HIV-infected individuals and uninfected CD4⁺ lymphocytes. Killing in all cases required T cell receptor (TCR)-mediated recognition or triggering. However, these CD8 cytotoxic T lymphocytes (CTL) killed HLA class I mismatched CD4* lymphocytes and CD4⁴ lymphocytes treated with a MoAb against HLA-A, B and C antigens (PA2.6) which blocks HLA class I-restricted killing. HLA class H-negativc CD4* T lymphoma cells (CEM.NK^R) were also killed by anti-CD3 inhibited CTL. Stimulation of peripheral blood lymphocytes (PBL) from HIV-infected individuals, but not uninfected controls, with concanavalin A (Con A) and IL-2, induced non-HLA-restricted TCR aft¹, CD8^f CTL which lysed CD4⁺ lymphocytes. Activation ofCD4’lymphocytes increased their susceptibility to CD8^f CTL-mediated lysis. In HIV infection, a population of non-HLA-restricted CTL which lyse activated CD4⁺ lymphocytes is expanded. The expansion of CTL with unusual characteristics is interesting, because the stimulus for this expansion is unknown. CTL which recognize activated CD4⁺ cells could play a role in immune regulation and the pathogenesis of A IDS.     

A natural cytokine mixture (IRX-2) and interference with immune suppression induce immune mobilization and regression of head and neck cancer

Prior studies indicate that combination immunotherapy of squamous cell cancer (SCC) of head and neck (H&N) with cytokines is feasible (Hadden et al., 1994). To induce immune regression of H&N SCC 20 stage II–IV patients received 3 weeks prior to surgery low dose cyclophosphamide (300 mg/M²), then 10 daily perilymphatic injections of a natural cytokine mixture (IRX-2) (150 units of IL-2 equivalence) and daily oral indomethacin and zinc.Tumorresponses, T-lymphocyte and subset counts, and toxicity were monitored. Six patients had major clinical responses (both complete [CR] and partial [PR]) without major toxicity. Five of 20 patients were lymphocytopenic (1242±88 mm³) prior to treatment and the immunotherapy induced marked significant increases in total lymphocyte counts, CD3+ T-cells, and both CD4+ and CD8+ T-cells as well as a population of CD3+, CD4−, and CD8− lymphocytes.The post treatment specimen of 18/20 patients showed histologically tumor fragmentation, overall reduction and diffuse infiltration with lymphocytes and plasma cells. Histologic tumor reductions in these patients averaged 44% and the lymphoid infiltration increased 4.7 fold from 9–42%. The immune infiltration of the tumor reflects varying degrees of both T- and B-cells and indicates immunization to the tumor. The immunization achieved may improve clinical control of H&N SCC by improving the possibility that surgical resection of advanced loco-regional disease will leave no viable tumor.     

Preferential loss of Leu 8−, CD45R−, HLA-DR+ CD8 cell subsets duringin vitro culture of mononuclear cells from human immunodeficiency virus type I (HIV)-seropositive former blood donors

Recent data from our laboratory showed that the CD4:CD8 cell ratio increased significantly duringin vitro culture of unstimulated mononuclear cells (MC) from HIV-infected persons. To test the hypothesis that this increase reflected a decline in CD8 cell levels, changes in CD4 and CD8 cell levels during culture of MC were assessed quantitatively. These analyses were accomplished using a Spectrum III flow cytometer, which analyzes a constant volume (0.02 ml) of cell suspension. The number of cells counted within this volume (termed the sip count) thus reflects the cell number in the suspension. To establish day 0 sip counts, aliquots consisting of 200,000 lymphocytes were treated with monoclonal antibodies, resuspended in 1 ml of buffer, and analyzed. Also on day 0, identical cell aliquots were placed in microtiter wells and cultured for 3 days. Cells retrieved from individual wells were then analyzed as on day 0. The mean relative recoveries (RR) of lymphocytes, CD4 cells, and CD8 cells were significantly lower in the HIV group (N=28) than in the control group (N=26). For the HIV group, CD8 cell RR was significantly lower than CD4 cell RR. Dualcolor analyses showed that CD4 cell loss in the HIV group did not occur preferentially within CD4 subsets defined by Leu 8 or CD45R expression. Similarly, CD8 cell loss did not occur preferentially within CD8 subsets defined by Leu 7 expression. In contrast, CD8 cell loss did preferentially affect Leu 8^–, CD45R^–, and HLA-DR⁺ CD8 subsets, compared to the reciprocal CD8 subsets. These findings indicate that some T cells from HIV-infected persons, particularly CD8 cells with phenotypes characteristic of activation, may not survive duringin vitro culture.     

Quantitative and functional analysis of a human lymphocyte subset with the T-helper (Leu 3/T 4+) phenotype and natural killer (NK)-cell characteristics in patients with malignancy

Approximately 20% of normal blood lymphocytes expressing the T-helper (Leu 3/T 4⁺) surface phenotype display natural killer (NK)-like features such as cytoplasmic granules and the ability to bind NK-cell targets. In this study, we have assessed the frequency, phenotypic features, and functional capabilities of such cells in a variety of lymphoid malignancies or solid tumors. In each patient group, the percentage of granular lymphocytes within the Leu 3/T 4⁺ T-helper subset was significantly increased. A large percentage of these cells coexpressed the Leu 7 or Leu 15 marker. When Leu 3⁺ cells from patients with high proportions of such NK-like cells (or Leu 3⁺-Leu 15⁺ cells from selected patients) were isolated with a fluorescence-activated cell sorter, these cells did not proliferate in response to allogeneic cells or T-cell mitogens, nor did they provide help for B-cell differentiation. They also did not suppress T-cell proliferative responses or B-cell differentiation. Freshly prepared Leu 3⁺ granular lymphocytes did not display NK-cell cytotoxic functions. However, after short-term culture in the presence of phytohemagglutinin (PHA), Leu 3⁺-Leu 15⁺ cells expressed T-cell growth factor (TCGF) receptors, had a detectable proliferative response to exogenous TCGF, and acquired the ability to lyse NK-cell targets. These studies demonstrate that, in a variety of malignancies, the lymphocyte subpopulation expressing the T-helper (Leu 3/T 4⁺) phenotype may be comprised largely of cells with NK-like features and functional capabilities distinct from those of classical helper T cells.     

Primary defect in CD8+ lymphocytes in the antibody deficiency disease (common variable immunodeficiency): abnormalities in intracellular production of interferon-gamma (IFN-γ) in CD28+ (‘cytotoxic’) and CD28− (‘suppressor’) CD8+ subsets

We have measured by flow cytometry the ability of subsets of CD8⁺ CD3⁺ lymphocytes within mononuclear cell preparations to make intracellular cytokines (IL-2, tumour necrosis factor-alpha (TNF-α) and IFN-γ) on stimulation in vitro with phorbol myristate acetate (PMA) and ionomycin for 16 h. These CD8⁺ subsets were defined by the presence or absence of CD28 or HLA-DR. Subsets of normal CD8⁺ cells were compared with cells from the antibody deficiency disease common variable immunodeficiency (CVID). In CVID there was a significant increase in the production of IFN-γ in the CD8⁺ CD28⁺ subset (‘cytotoxic’). This reflects a shift in this disease towards an excessive Th1 response away from B cell help. Paradoxically, some CVID patients also showed a reduction in IFN-γ production in the CD8⁺ CD28^? subset (‘suppressor’) which was associated with a failure of these cells to maintain a state of activation after a stimulus in vitro. The B cell problem in this disease is known to be related to a failure of T cell help shown by an inability to produce the antigen-specific CD4⁺ memory T cells needed for successful B cell maturation. The two pathological CD28 subsets of CD8⁺ cells we have found in CVID may both be detrimental to a normal CD4-dependent immune response. The CD28^? suppressor subset expands and is unable to maintain activation and cytokine secretion, and the CD28⁺ cytotoxic subset is over-producing the Th1 cytokine IFN-γ.     

Mouse pancreatic beta cells express MHC class II and stimulate CD4+ T cells to proliferate

Type 1 diabetes results from destruction of pancreatic beta cells by autoreactive T cells. Both CD4⁺ and CD8⁺ T cells have been shown to mediate beta‐cell killing. While CD8⁺ T cells can directly recognize MHC class I on beta cells, the interaction between CD4⁺ T cells and beta cells remains unclear. Genetic association studies have strongly implicated HLA‐DQ alleles in human type 1 diabetes. Here we studied MHC class II expression on beta cells in nonobese diabetic mice that were induced to develop diabetes by diabetogenic CD4⁺ T cells with T‐cell receptors that recognize beta‐cell antigens. Acute infiltration of CD4⁺ T cells in islets occurred with rapid onset of diabetes. Beta cells from islets with immune infiltration expressed MHC class II mRNA and protein. Exposure of beta cells to IFN‐γ increased MHC class II gene expression, and blocking IFN‐γ signaling in beta cells inhibited MHC class II upregulation. IFN‐γ also increased HLA‐DR expression in human islets. MHC class II⁺ beta cells stimulated the proliferation of beta‐cell‐specific CD4⁺ T cells. Our study indicates that MHC class II molecules may play an important role in beta‐cell interaction with CD4⁺ T cells in the development of type 1 diabetes.     

Tolerogen-induced interferon-producing killer dendritic cells (IKDCs) protect against EAE

Natural killer (NK) cells and dendritic cells (DCs) have been shown to link the innate and adaptive immune systems. Likewise, a new innate cell subset, interferon-producing killer DCs (IKDCs), shares phenotypic and functional characteristics with both DCs and NK cells. Here, we show IKDCs play an essential role in the resolution of experimental autoimmune encephalomyelitis (EAE) upon treatment with the tolerizing agent, myelin oligodendrocyte glycoprotein (MOG), genetically fused to reovirus protein σ1 (termed MOG-pσ1). Activated IKDCs were recruited subsequent MOG-pσ1 treatment of EAE, and disease resolution was abated upon NK1.1 cell depletion. These IKDCs were able to kill activated CD4⁺ T cells and mature dendritic DCs, thus, contributing to EAE remission. In addition, IKDCs were responsible for MOG-pσ1-mediated MOG-specific regulatory T cell recruitment to the CNS. The IKDCs induced by MOG-pσ1 expressed elevated levels of HVEM for interactions with cognate ligand-positive cells: LIGHT⁺ NK and T_eff cells and BTLA⁺ B cells. Further characterization revealed these activated IKDCs being MHC class II^high, and upon their adoptive transfer (CD11c⁺NK1.1⁺MHC class II^high), IKDCs, but not CD11c⁺NK1.1⁺MHC class II^{intermediate/low} (unactivated) cells, conferred protection against EAE. These activated IKDCs showed enhanced CD107a, PD-L1, and granzyme B expression and could present OVA, unlike unactivated IKDCs. Thus, these results demonstrate the interventional potency induced HVEM⁺ IKDCs to resolve autoimmune disease.     

Tumor necrosis factor production by human T-cells stimulated with bacterial superantigens

Tumor necrosis factor (TNF) production from T-cells stimulated with superantigenic exotoxins, staphylococcal enterotoxin B and streptococcal pyrogenic exotoxin A was investigated in the presence of cells bearing distinct isotypes of HLA class II molecules. The main T-cell subset for TNF production was investigated in parallel. Similarly high levels of TNF production were induced upon stimulation with the toxins in the presence of DR⁺ or DQ⁺ cells, but only marginal levels of TNF production were induced in the presence of DP⁺ cells. Although both CD4⁺ T-cells and CD8⁺ T-cells produced TNF-α and TNF-β in response to toxin stimulation in the presence of HLA class II⁺ cells, the former T-cell subset was the major source of producers of TNF-α and TNF-β.     

Lymphocyte subsets in distinct lung compartments show a different ability to produce interferon-gamma (IFN-γ) during a pulmonary immune response

Lymphocytes play an important immunoregulatory role in pulmonary immune responses. By releasing cytokines they can control the cell–cell communication of other participating cells. Although it is well established that the lung lymphocytes, localized in distinct compartments, differ in their subset composition, little is known about cytokine production in these compartments during immune responses. Lewis rats were immunized by intravenous administration of sheep erythrocytes on day 0 and day 7 and challenged intratracheally with sheep erythrocytes on day 10. Four days after intratracheal (i.t.) challenge the composition of lymphocyte subsets (CD2⁺, CD4⁺, CD8⁺, B cells, natural killer (NK) cells) in the spleen, blood, lung perfusate, lung tissue and bronchoalveolar lavage fluid (BALF) was characterized, and intracellular IFN-γ was detected in these subsets by flow cytometry. Comparing control and immunized animals, no changes were found in lymphocyte numbers, subsets or the percentage of IFN-γ-producing lymphocytes in the spleen, blood and lung perfusate. In lung tissue and BALF, however, the absolute number of all lymphocyte subsets and the percentage of IFN-γ-producing lymphocytes were increased. When the lymphocyte subsets were analysed an increased percentage of IFN-γ-producing T cells was found in lung tissue (4.5 ± 0.6% versus 12.8 ± 1.1%) and in BALF (7.8 ± 1.4% versus 14.8 ± 1.9%) of immunized animals opposed to controls, this increase being seen in both CD4⁺ and CD8⁺ cells. Thus, there is an accumulation of T cells with an increased potential to produce IFN-γ in the lung interstitium and the bronchoalveolar space during pulmonary immune responses.     

CD127 expression and regulation are altered in the memory CD8 T cells of HIV-infected patients--reversal by highly active anti-retroviral therapy (HAART)

HIV infection activates abnormally the immune system and the chronic phase is accompanied by marked alterations in the CD8 compartment. The expression of CD127 (IL-7R alpha chain) by memory CD8 T lymphocytes in HIV-infected patients is analysed and reported. The memory CD8 T cell subset was characterized by expression of CD45RA and CD27 markers, and CD127 cell surface expression was measured ex vivo by four-colour flow cytometry. HIV infection was associated with a fall in the proportion of CD127(+) cells among memory CD8 lymphocytes that resulted in a higher CD127(-) CD45RA(-)CD27(+) CD8 T cell count in HIV-infected patients. Diminished CD127 cell surface expression [mean fluorescence intensity (MFI)] by positive cells was also observed in this subset. The data suggest that these defects were reversed by highly active anti-retroviral therapy (HAART). The regulation of CD127 expression was also studied in vitro. Down-regulation of CD127 by interleukin (IL)-7 was observed in memory CD8 lymphocytes from healthy donors and HAART patients. Expression of CD127 by memory CD8 lymphocytes cultured in the absence of IL-7 confirmed that IL-7R regulation is altered in viraemic patients. Under the same experimental conditions, memory CD8 lymphocytes from HAART patients were shown to express CD127 at levels comparable to cells from healthy individuals. Altered CD127 cell surface expression and defective CD127 regulation in the memory CD8 T lymphocytes of HIV-infected patients are potential mechanisms by which these cells may be impeded in their physiological response to endogenous IL-7 stimulatory signals. Our data suggest that these defects are reversed during the immune reconstitution that follows HAART.     

CD8high(CD57+) T cells in normal,healthy individuals specifically suppress the generation of cytotoxic T lymphocytes to Epstein-Barr virus-transformed B cell lines

We have previously identified two subsets of CD8⁺, CD57⁺ lymphocytes in normal peripheral blood: i) T cells expressing high levels [CD8^high(CD57⁺)] and ii) natural killer cells expressing low levels of surface CD8 [CD8^low(CD57⁺)]. We investigated the cytotoxic and suppressive function of CD8^high(CD57⁺) T lymphocytes from normal, healthy individuals using standard chromium-release assays and limiting dilution analysis. In normal, healthy subjects, this cell subset suppressed the generation of cytotoxic T lymphocytes (CTL) to autologous, Epstein-Barr virus (EBV)-transformed B cell lines (BCL). Depletion of CD8^high(CD57⁺) T lymphocytes from peripheral blood mononuclear cells (PBMC) resulted in a three- to sevenfold rise in CTL precursor frequency to autologous EBV-transformed BCL, but not allogeneic PBMC or BCL by LDA. Replacement of CD8^high(CD57⁺) T lymphocytes in limiting dilution cultures led to the dose-dependent suppression of EBV-specific, but not allogeneic, CTL generation. Supernatant from CD8^high(CD57⁺) T lymphocytes cultured with autologous BCL did not exhibit suppression, suggesting that soluble factors were not responsible. As CD8^high(CD57⁺) T lymphocytes did not, themselves, exhibit cytotoxicity against autologous BCL, removal of BCL stimulator cells in co-culture was not the mechanism of suppression. Furthermore, while the CD8^high(CD57⁺) T lymphocytes from healthy subjects suppressed the generation of CTL to autologous BCL, they did not suppress the cytotoxic activity of established mixed lymphocyte reactions or peptide-specific CTL clones, as has been reported in bone marrow transplant recipients and human immunodeficiency virus patients. This suggests that CD8^high(CD57⁺) T lymphocytes from healthy subjects suppress the generation of, rather than killing by, CTL in a contact-dependent manner. To our knowledge, this is the first identification of a phenotypically distinct subset of human CD8⁺ T cells that can suppress generation of antigen-specific major histocompatibility complex class I-restricted CTL.     

Patterns of major histocompatibility complex class II expression by T cell subsets in different immunological compartments. 2. Altered expression and cell function following activation in vivo

This study characterizes antigen-induced phenotypic and functional aspects of major histocompatibility complex (MHC) class II expression on recirculating T cells in efferent lymph. In vivo secondary, but not primary challenge is associated with both kinetic and phenotypic alterations in class II expression by T cells. All three major T cell subsets, CD4⁺, CD8⁺ and T19⁺ (γδ T cell receptor), show an approximate four fold increase in the level of MHC class II expression during secondary responses. No changes in B cell expression of class II were seen. Resting efferent lymph T cells are predominantly either class II^? or DR⁺DQ^? but this changes to DR⁺DQ⁺ after antigenic challenge. The antigen-presenting function of these class II⁺ T cells was investigated at daily intervals after in vivo antigenic challenge. T cells from non-activated lymph nodes could not induce proliferation of antigen-specific T cells with soluble antigen but were weakly stimulatory in allo-mixed lymphocyte reaction (MLR) at high (> 2:1) stimulator cell ratios. Activated T cells isolated during secondary in vivo responses, and expressing increased quantities of MHC class II, were positive stimulator cells in the MLR. In contrast these cells could not present soluble antigen or trypsin-digested antigen to the T cell lines. In the MLR assays, the relative stimulation by class II⁺ T cells correlates with the levels of class II expression. We conclude from these experiments that both quantitative and qualitative changes in MHC class II, induced on T cells under physiological conditions, play a role in the regulation of the immune response in vivo but that that role is not simply one of presentation of soluble antigen.     

An Examination of the Development and Localization of Key Immune Cells in Developing Pouch Young of the Red-Tailed Phascogale (Phascogale calura)

Cells expressing the surface markers CD3, CD4, CD79b, IgM, MHC class II, and ModoUG (nonclassical MHC class I) were detected in red-tailed phascogale tissues using immunohistochemistry, and the appearance and localization of cells observed here was consistent with previous observations in other marsupial species. CD3⁺ cells were first detected at one day postpartum (dpp) in the thymus, followed by ModoUG⁺ cells at 5–7 dpp in the thymus and lymph nodes. CD79b⁺ cells were first detected at 12–14 dpp in bone marrow, spleen, and lymph nodes. IgM⁺ cells were first detected at 12–14 dpp in thymus, bone marrow, spleen, and lymph nodes. MHC class II⁺ cells were first detected at 12–14 dpp in thymus, bone marrow, and lymph nodes. CD4⁺ cells were detected in adult thymus and spleen only. The presence of the mature immune cell populations and their localization to characteristic T and B cell zones in mature lymphoid tissues with normal histological structure indicates that red-tailed phascogales develop immunocompetence by the end of pouch life. Anat Rec, 302:1985–2002, 2019. © 2019 American Association for Anatomy     

Leucocyte phenotypes in involuting and fully involuted mammary glandular tissues and secretions of sheep

Mammary glandular tissues and mammary secretions were obtained from sheep at 2–60 d after weaning to study the leucocyte phenotypes associated with mammary involution. From 2–4 d after weaning, neutrophils were the predominant leucocytes in the alveolar and ductal lumina. Lymphocytes were present in the alveolar and ductal epithelium, interalveolar and periductal areas. Most of the lymphocytes in the alveolar and ductal epithelium (IEL) were CD8⁺, some were CD45R⁺ and few were CD4⁺. In the periductal clusters and in the interalveolar areas most of the lymphocytes were CD4⁺. There was a significant increase (P < 0.05) in the percentages of CD45R⁺ granulated IEL from 2 to 7 d after weaning, and this paralleled the increase in the percentages of apoptotic cells in the glandular epithelium. By 7–60 d after weaning, most cells within the alveolar and ductal lumina were macrophages followed by predominantly CD8⁺ lymphocytes. CD8⁺ lymphocytes were still predominant in the alveolar and ductal epithelium while CD4⁺ cells were predominant in the interalveolar areas. Very few γδ⁺ T cells were observed at all the stages examined. The cells in the mammary secretions correlated with those observed in the alveolar and ductal lumina. At the early stages of involution, the neutrophils and macrophages were heavily laden with lipid droplets, casein and cellular debris. The most interesting feature was the presence of cells either with extensive cytoplasmic processes (LCA⁺ MHC class II⁺) or cytoplasmic veils (LCA⁺ MHC class II⁺CD1⁺), probably dendritic cells. It is concluded that the cellular constituents of the mammary gland at the latter part of involution may afford the mammary gland more resistance to infection than the lactating gland and the gland at early stages of involution. The CD45R⁺ IEL may trigger apoptotic cell death in the mammary glandular epithelium during mammary involution.     

Correlation of the percentage of activated,CD3+DR+ lymphocytes to serum neopterin level in HIV-seropositive haemophiliacs

Summary The percentage of activated, CD3+ DR+ and CD8+Leu7+ lymphocytes and the serum neopterin concentration were determined in 17 HIV-seropositive and 10 HIV-seronegative haemophiliacs and in 11 healthy control subjects. All three parameters tested were found to be significantly higher in the seropositive patients than in the seronegative controls. In the seropositive group, a significant positive correlation was found between the neopterin levels and the percentage of CD3+DR+ cells. By contrast, no significant negative or positive correlation was observed between the neopterin levels and the percentage of the CD8+Leu7+ subset. These data suggest that in the HIV-infected patients the activated T cells responsible for the stimulation of macrophages to produce neopterin are those that do not carry CD8.Abbreviations CD cluster designation - AIDS acquired immune deficiency syndrome - HIV human immunodeficiency virus - CDC Centers for Disease Control     

DC‐induced CD8+ T‐cell response is inhibited by MHC class II‐dependent DX5+CD4+ Treg

CD4⁺ T cells are important for CD8⁺ T‐cell priming by providing cognate signals for DC maturation. We analyzed the capacity of CD4⁺ T cells to influence CD8⁺ T‐cell responses induced by activated DC. Surprisingly, mice depleted for CD4⁺ cells were able to generate stronger antigen‐specific CD8⁺ T‐cell responses after DC vaccination than non‐depleted mice. The same observation was made when mice were vaccinated with MHC class II^?/? DC, indicating the presence of a MHC class II‐dependent CD4⁺ T‐cell population inhibiting CD8⁺ T‐cell responses. Recently we described the expansion of DX5⁺CD4⁺ T cells, a T‐cell population displaying immune regulatory properties, upon vaccination with DC. Intriguingly, we now observe an inverse correlation between CD8⁺ T‐cell induction and expansion of DX5⁺CD4⁺ T cells as the latter cells did not expand after vaccination with MHC class II^?/? DC. In vitro, DX5⁺CD4⁺ T cells were able to limit proliferation, modulate cytokine production and induce Foxp3⁺ expression in OVA‐specific CD8⁺ T cells. Together, our data show an inhibitory role of CD4⁺ T cells on the induction of CD8⁺ T‐cell responses by activated DC and indicate the involvement of DX5⁺CD4⁺, but not CD4⁺CD25⁺, T cells in this process.