HIV-gp120 can block CD4-class II MHC-mediated adhesion

A possible component of the immune dysfunction associated with infection by HIV is the inhibition of CD4 function resulting from the avid binding of soluble HIV envelope glycoprotein (gp120) to cell surface CD4. We assessed CD4 function by measuring the ability of CD4+ T cells to form conjugates with cell size lipid vesicles, artificial target cells (ATC), bearing the natural ligand for CD4, MHC class II proteins. Conjugate formation was a transient process with the greatest number of specific cell to ATC conjugates found after approximately 30 min of incubation at 37 degrees C. Addition of gp120 specifically blocked conjugates between CD4+ cells and class II ATC in a concentration-dependent manner. These data indicate that T lymphocyte adhesion mediated by CD4 is a dynamic event and that binding of gp120 to CD4 is able to disrupt the normal progression of the interaction between CD4+ T lymphocytes and class II+ APC.     

Costimulation of Naive CD8+ Lymphocytes Induces CD4 Expression and Allows Human Immunodeficiency Virus Type 1 Infection

Human immunodeficiency virus type 1 (HIV-1) infection requires cell surface expression of CD4. Costimulation of CD8⁺/CD4⁻ T lymphocytes by anti-CD3 and anti-CD28 antibodies or by allogeneic dendritic cells induced expression of CD4 and rendered these CD8 cells susceptible to HIV-1 infection. Naive CD45RA⁺ cells responded with greater expression of CD4 than did CD45RO⁺ cells. CD8⁺ lymphocytes derived from fetal or newborn sources exhibited a greater tendency to express CD4, consistent with their naive states. This mechanism of infection suggests HIV-induced perturbation of the CD8 arm of the immune response and could explain the generally rapid disease progression seen in HIV-infected children.     

Regulation of immunoglobulin synthesis by human T cell subsets as defined by anti-D44 monoclonal antibody within the CD4+ and CD8+ subpopulations

In our previous paper, we demonstrated that anti-D44 MAb can, in the presence of complement, eliminate all the allocytotoxicity generated during a mixed lymphocyte reaction without affecting the alloproliferative response. As approximately 70% of CD4+ cells and 30% of CD8+ will be stained with anti-D44 MAb, we researched the functional role of the D44+ and D44- cells in each of these T cell subsets in the PWM-induced antibody response. We found that most of the helper activity for immunoglobulin (Ig) synthesis was mediated by CD4+ D44+ lymphocytes and that virtually all the suppressive activity was mediated by CD8+ D44- lymphocytes. Surprisingly enough, we noticed that the low level of Ig synthesis induced in B cells by CD4+ D44- lymphocytes could be strongly amplified by the addition of radiosensitive CD8+ lymphocytes, suggesting coexisting opposite immunoregulatory functions within the CD8+ T cell subset. These results, together with previous data, indicate that anti-D44 MAb subdivides T cells into subpopulations with distinct functional repertoires: a CD4+ D44+ helper subpopulation, a CD8+ D44+ cytotoxic subpopulation, and a CD8+ D44- suppressor subpopulation.     

Alteration of circadian rhythmicity of CD3+CD4+ lymphocyte subpopulation in healthy aging

The CD4+ T helper/inducer and the CD8+ T suppressor/cytotoxic are major lymphocyte subsets that play a key role in cell-mediated immunity. Aging-related changes of immune function have been demonstrated. The purpose of this study is to analyze the dynamics of variation of these specific lymphocyte subsets in the elderly. In our study cortisol and melatonin serum levels were measured and lymphocyte subpopulation analyses were performed on blood samples collected every four hours for 24 hours from fifteen healthy young middle-aged subjects (age range 36-55 years) and fifteen healthy elderly male subjects (age range 67-79 years). A clear circadian rhythm was validated for the time-qualified changes of CD3+ and CD4+ cells with acrophase at night and for the time-qualified changes of CD8+ cells with acrophase at noon in young middle-aged subjects and for the time-qualified changes of CD3+ cells with acrophase at night and for the time-qualified changes of CD8+ cells with acrophase at noon in elderly subjects. No clear circadian rhythm was validated for the time-qualified changes of CD4+ cells in elderly subjects. No statistically significant correlation among lymphocyte subsets was found in elderly subjects. In elderly subjects CD3+ lymphocyte percentage was higher in the photoperiod and in the scotoperiod and cortisol serum level were higher in the scotoperiod in respect to young middle-aged subjects. In the elderly there is an alteration of circadian rhythmicity of T helper/inducer lymphocytes and this phenomenon might contribute to the aging-related changes of immune responses.     

Developmental regulation of αβ T cell antigen receptor assembly in immature CD4+CD8+ thymocytes

Most lymphocytes of the T cell lineage develop along the CD4/CD8 pathway and express antigen receptors on their surfaces consisting of clonotypic αβ chains associated with invariant CD3-γδε components and ζ chains, collectively referred to as the T cell antigen receptor complex (TCR). Expression of the TCR complex is dynamically regulated during T cell development, with immature CD4⁺CD8⁺ thymocytes expressing only 10% of the number of αβ TCR complexes on their surfaces expressed by mature CD4⁺ and CD8⁺ T cells. Recent evidence demonstrates that low surface TCR density on CD4⁺CD8⁺ thymocytes results from the limited survival of a single TCR component within the ER, the TCRα chain, which has a half life of only 15 minutes in immature thymocytes, compared to >75 minutes in mature T cells. Instability of TCRα proteins in immature CD4⁺CD8⁺ thymocytes represents a novel mechanism by which expression of the multisubunit TCR complex is quantitatively regulated during T cell development. In the current review we discuss our recent findings concerning the assembly, intracellular transport, and expression of αβ TCR complexes in CD4⁺CD8⁺ thymocytes and comment on the functional significance of TCRα instability during T cell development.     

Age‐associated alterations in CD8α+ dendritic cells impair CD8 T‐cell expansion in response to an intracellular bacterium

Age‐associated decline in immunity to infection has been documented across multiple pathogens, yet the relative contributions of the aged priming environment and of lymphocyte‐intrinsic defects remain unclear. To address the impact of the aging environment on T‐cell priming, adult naïve OT‐I TCR transgenic CD8 T cells, specific for the H‐2K^b‐restricted immunodominant OVA_257‐264 epitope, were transferred into adult or old recipient mice infected with the recombinant intracellular bacterium Listeria monocytogenes carrying the chicken ovalbumin protein (Lm‐OVA). We consistently found that adult OT‐I CD8 expansion was reduced in aged recipient mice, and this correlated with numeric, phenotypic, and functional defects selectively affecting CD8α+ dendritic cells (DC). Following Lm‐OVA infection, aged mice failed to accumulate CD8α+ DC in the spleen, and these cells expressed much lower levels of critical costimulatory molecules in the first three days following infection. Further, aged CD8α+ DC showed impaired uptake of the bacteria at very early time points following infection. Treatment of aged mice with Flt3 ligand (Flt3L) improved the number of DC present in the spleen prior to Lm‐OVA infection, and improved, but did not reconstitute, OT‐I expansion to Lm‐OVA infection. These results suggest that age‐associated changes in antigen uptake, pathogen sensing, and/or antigen presentation contribute to impaired adaptive immune responses to microbial pathogens with aging.     

Effect of HIF1α on Foxp3 expression in CD4+CD25− T lymphocytes

The aim of the present study was to investigate the effect of HIF1α on Foxp3 expression in CD4⁺CD25^? T lymphocytes. CD4⁺CD25^? T lymphocytes were sorted from PBMC using a CD4⁺CD25⁺ regulatory T cell isolation kit. Lentivirus containing lentiviral vector that overexpressed HIF1α (HIF‐lenti) and those containing empty expression vector (control‐lenti) were produced. Meanwhile, lentivirus that contained lentiviral vector that suppressed HIF1α expression (siHIF‐lenti) and those containing control vector (sicontrol‐lenti) were also generated. The sorted CD4⁺CD25^? T lymphocytes were infected with HIF‐lenti, control‐lenti, siHIF‐lenti, and sicontrol‐lenti, respectively. Approximately 72 hr after transduction, real‐time PCR and Western blot were carried out to analyze the RNA and protein expression level of HIF1α and Foxp3. CD4⁺CD25^? T lymphocytes cultured under 21% O₂, 5% CO₂ (normoxia) and 1% O₂, 5% CO₂ (hypoxia) were used as control. Our results showed that overexpression of HIF1α increased both mRNA and protein expression of Foxp3 and, meanwhile, suppression of HIF1α expression by RNAi could reverse high Foxp3 expression in CD4⁺CD25^? T lymphocytes caused by hypoxic culture. These results suggested that hypoxia could stimulate Foxp3 expression by increasing HIF1α expression in CD4⁺ T lymphocytes which may promote CD4⁺ T lymphocytes to convert to Treg.

     

Functional CD8+ T cell responses in lethal Ebola virus infection

Ebola virus (EBOV) causes highly lethal hemorrhagic fever that leads to death in up to 90% of infected humans. Like many other infections, EBOV induces massive lymphocyte apoptosis, which is thought to prevent the development of a functional adaptive immune response. In a lethal mouse model of EBOV infection, we show that there is an increase in expression of the activation/maturation marker CD44 in CD4(+) and CD8(+) T cells late in infection, preceding a dramatic rebound of lymphocyte numbers in the blood. Furthermore, we observed both lymphoblasts and apoptotic lymphocytes in spleen late in infection, suggesting that there is lymphocyte activation despite substantial bystander apoptosis. To test whether these activated lymphocytes were functional, we performed adoptive transfer studies. Whole splenocytes from moribund day 7 EBOV-infected animals protected naive animals from EBOV, but not Marburgvirus, challenge. In addition, we observed EBOV-specific CD8(+) T cell IFN-gamma responses in moribund day 7 EBOV-infected mice, and adoptive transfer of CD8(+) T cells alone from day 7 mice could confer protection to EBOV-challenged naive mice. Furthermore, CD8(+) cells from day 7, but not day 0, mice proliferated after transfer to infected recipients. Therefore, despite significant lymphocyte apoptosis, a functional and specific, albeit insufficient, adaptive immune response is made in lethal EBOV infection and is protective upon transfer to naive infected recipients. These findings should cause a change in the current view of the 'impaired' immune response to EBOV challenge and may help spark new therapeutic strategies to control lethal filovirus disease.     

Characterization of porcine T lymphocytes and their immune response against viral antigens.

T lymphocytes play a central role in the antigen-specific immune response against various pathogens. To detect and to characterize porcine T lymphocytes, monoclonal antibodies (mAb) against leukocyte differentiation antigens had been raised and classified for their specificity. Analyses of porcine T lymphocytes with specific mAb against CD4 and CD8 differentiation antigens revealed differences in the composition of the porcine T-lymphocyte population compared to other species. In addition to the known subpopulations, CD4+CD8- T helper cells and CD4-CD8+ cytolytic T lymphocytes, extra-thymic CD4+CD8+ T lymphocytes and a substantial proportion of CD2-CD4-CD8- T cell receptor (TcR)-gamma delta+ T cells could be detected in swine. Functional analyses of porcine T-lymphocyte subpopulations revealed the existence of two T-helper cell fractions with the phenotype CD4+CD8- and CD4+CD8+. Both were reactive in primary immune responses in vitro, whereas only cells derived from the CD4+CD8+ T-helper-cell subpopulation were able to respond to recall antigen in a secondary immune response. With regard to T lymphocytes with cytolytic activities, two subsets within the CD4-CD8+ T-cell subpopulation could be defined by the expression of CD6 differentiation antigens: CD6- cells which showed spontaneous cytolytic activity and CD6+ MHC I-restricted cytolytic T lymphocytes including virus-specific cytolytic T lymphocytes. These results enable now a detailed view into the porcine T-cell population and the reactivity of specific T cells involved in the porcine immune response against pathogens. Furthermore this knowledge offers the possibility to investigate specific interactions of porcine T lymphocytes with virus-specific epitopes during vaccination and viral infections.     

Comodulation of CD3 and CD4. Evidence for a specific association between CD4 and approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes

The aggregation of a specific class of lymphocyte surface molecules results in patching, capping, and surface modulation of the aggregated ligand. Both CD4, an associative recognition structure found on helper T lymphocytes, and CD3, a component of the T cell receptor complex, are members of this functional subgroup. When 125I-labeled monoclonal antibodies reactive with either CD4 (19Thy 5D7) or CD3 (RW24B6) were bound to T lymphocytes, the subsequent addition of goat anti-mouse Ig resulted in their rapid, temperature-dependent internalization. Whereas the binding of 125I-19Thy 5D7 (anti-CD4) was inhibited by greater than 90% in the presence of unlabeled 19Thy 5D7, no inhibition occurred in the presence of unlabeled antibody reactive with CD3 (RW28C8). We took advantage of the fact that these antibodies were of different isotypes (19Thy 5D7:IgG2a; RW28C8:IgGl) to determine whether the internalization of CD3 induced the comodulation of CD4. T lymphocytes preincubated with 125I-19Thy5D7 (anti-CD4) and unlabeled RA28C8 (anti-CD3) were treated with goat anti-mouse IgGl under conditions shown to quantitatively internalize CD3. After 1 h at 37 degrees C, T lymphocytes had internalized 10.5 +/- 2.6% (n = 3) of their antibody-bound cell surface CD4. After similar incubations with media alone or with goat anti-mouse IgGl in the absence of prebound RW28C8 (anti-CD3), no internalization of CD4 could be detected. Control antibodies reactive with CD45R (2H4, IgGl) also failed to induce the internalization of CD4. Similar results were obtained by using a helper T cell clone (T4C1) that internalized 9.6 +/- 2.8% (n = 3) of its antibody-bound cell surface CD4 in response to CD3 modulation. In a reciprocal experiment, 125I-anti-CD3 (RW24B6, IgG2b) was preincubated with T4Cl cells together with unlabeled anti-CD4 (12T4D11, IgG1) prior to the addition of goat anti-mouse IgGl. The quantitative modulation of CD4 induced the co-internalization of 4.6 +/- 0.6% (n = 3) of cell surface CD3. These results suggest that approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes are specifically associated with CD4. Furthermore, our results suggest that an average of two CD4 molecules associate with each CD3:T cell receptor complex.     

Sepsis-induced apoptosis causes progressive profound depletion of B and CD4+ T lymphocytes in humans

Patients with sepsis have impaired host defenses that contribute to the lethality of the disorder. Recent work implicates lymphocyte apoptosis as a potential factor in the immunosuppression of sepsis. If lymphocyte apoptosis is an important mechanism, specific subsets of lymphocytes may be more vulnerable. A prospective study of lymphocyte cell typing and apoptosis was conducted in spleens from 27 patients with sepsis and 25 patients with trauma. Spleens from 16 critically ill nonseptic (3 prospective and 13 retrospective) patients were also evaluated. Immunohistochemical staining showed a caspase-9-mediated profound progressive loss of B and CD4 T helper cells in sepsis. Interestingly, sepsis did not decrease CD8 T or NK cells. Although there was no overall effect on lymphocytes from critically ill nonseptic patients (considered as a group), certain individual patients did exhibit significant loss of B and CD4 T cells. The loss of B and CD4 T cells in sepsis is especially significant because it occurs during life-threatening infection, a state in which massive lymphocyte clonal expansion should exist. Mitochondria-dependent lymphocyte apoptosis may contribute to the immunosuppression in sepsis by decreasing the number of immune effector cells. Similar loss of lymphocytes may be occurring in critically ill patients with other disorders.     

The CD160+ CD8high cytotoxic T cell subset correlates with response to HAART in HIV-1+ patients

We investigated the circulating cytotoxic CD160+ CD8(high) subset in correlation to antiviral immunity and response to highly active antiretroviral therapy (HAART) in HIV+ subjects. The study included 45 treatment-naive patients receiving HAART for 18 months, retrospectively defined as good (n=29) and transient (n=16) responders. HIV-specific CD8 T lymphocyte levels were measured by IFNgamma production in response to p17 Gag, in the presence of immobilized anti-CD160 mAb. We report a significantly increased baseline level of CD160+ CD8(high) subset in good therapy responders. CD160+ CD8(high) subset correlates with CD4+ T cell count, immune activation, and viral load. CD160+ CD8(high) lymphocytes contain a high amount of Granzyme B and include virus-specific T lymphocytes in HIV-1+ subjects. Co-stimulation through CD160 molecules enhances IFNgamma production in response to p17 Gag. Therefore, the CD160+ CD8(high) subset may be useful for monitoring of virus-specific cellular immunity and predicting response to antiretroviral therapy in chronic HIV-1 infection.     

Human T lymphocytes expressing the C3b/C4b complement receptor type one (CR1, CD35) belong to Fc gamma receptor-positive CD4-positive T cells

The phenotypic characteristics of human T lymphocytes expressing the C3b/C4b complement receptor type one (CR1, CD35) were investigated using dual-color surface immunofluorescence and cytofluorometric analysis of stained peripheral blood mononuclear cells (PBMC) from normal individuals. Two to ten percent of PBMC coexpressed CR1 and the CD5, CD2, or CD3 antigen. CR1 was detected on a subset of CD4+ T lymphocytes but not on CD8+ or on Leu-7+ lymphocytes. Costaining for CR1 and for the CD4 subpopulation markers anti-Leu-8, TQ1, OKT17, 2H4, and 4B4 indicated that CR1 on lymphocytes may be coexpressed with any of these phenotypic determinants. All CR1+ lymphocytes expressed Fc gamma receptors (Fc gamma Rs) as assessed by their ability to bind biotinylated dimeric human IgG. The expression of CR1 was increased in mixed lymphocyte reaction with kinetics similar to those of HLA-DR antigen expression. Coexpression of CR1 and Fc gamma R+ may provide a subset of CD4+ lymphocytes with an enhanced ability to bind and respond to C3-bearing complexes of IgG and antigen.     

Requirement for CD40 Ligand, CD4+ T Cells, and B Cells in an Infectious Mononucleosis-Like Syndrome

Respiratory challenge with the murine gammaherpesvirus 68 (gammaHV-68) results in productive infection of the lung, the establishment of latency in B lymphocytes and other cell types, transient splenomegaly, and prolonged clonal expansion of activated CD8(+) CD62L(lo) T cells, particularly a Vbeta4(+) CD8(+) population that is found in mice with different major histocompatibility complex (MHC) haplotypes. Aspects of the CD8(+)-T-cell response are substantially modified in mice that lack B cells, CD4(+) T cells, or the CD40 ligand (CD40L). The B-cell-deficient mice show no increase in Vbeta4(+) CD8(+) T cells. Similar abrogation of the Vbeta4(+) CD8(+) response is seen following antibody-mediated depletion of the CD4(+) subset, through the numbers of CD8(+) CD62L(lo) cells are still significantly elevated. Virus-specific CD4(+)-T-cell frequencies are minimal in the CD40L(-/-) mice, and the Vbeta4(+) CD8(+) population remains unexpanded. Apparently B-cell-CD4(+)-T-cell interactions play a part in the gammaHV-68 induction of both splenomegaly and non-MHC-restricted Vbeta4(+) CD8(+)-T-cell expansion.     

The versatility of the αβ T‐cell antigen receptor

The T‐cell antigen receptor is a heterodimeric αβ protein (TCR) expressed on the surface of T‐lymphocytes, with each chain of the TCR comprising three complementarity‐determining regions (CDRs) that collectively form the antigen‐binding site. Unlike antibodies, which are closely related proteins that recognize intact protein antigens, TCRs classically bind, via their CDR loops, to peptides (p) that are presented by molecules of the major histocompatibility complex (MHC). This TCR‐pMHC interaction is crucially important in cell‐mediated immunity, with the specificity in the cellular immune response being attributable to MHC polymorphism, an extensive TCR repertoire and a variable peptide cargo. The ensuing structural and biophysical studies within the TCR‐pMHC axis have been highly informative in understanding the fundamental events that underpin protective immunity and dysfunctional T‐cell responses that occur during autoimmunity. In addition, TCRs can recognize the CD1 family, a family of MHC‐related molecules that instead of presenting peptides are ideally suited to bind lipid‐based antigens. Structural studies within the CD1‐lipid antigen system are beginning to inform us how lipid antigens are specifically presented by CD1, and how such CD1‐lipid antigen complexes are recognized by the TCR. Moreover, it has recently been shown that certain TCRs can bind to vitamin B based metabolites that are bound to an MHC‐like molecule termed MR1. Thus, TCRs can recognize peptides, lipids, and small molecule metabolites, and here we review the basic principles underpinning this versatile and fascinating receptor recognition system that is vital to a host's survival.     

Differential Targeting of Viral Components by CD4+ versus CD8+ T Lymphocytes in Dengue Virus Infection

Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4⁺ and CD8⁺ T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4⁺ and 21 are CD8⁺ T cell epitopes. We observe that whereas CD8⁺ T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4⁺ epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4⁺ T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4⁺ and CD8⁺ T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.     

Essential role for CD40 ligand interactions in T lymphocyte-mediated modulation of the murine immune response to pneumococcal capsular polysaccharides

Protection against infection with pneumococci is provided by anti-capsular polysaccharide (caps-PS) Abs. We investigated whether CD40 ligand (CD40L) plays a role in T lymphocyte-mediated regulation of the immune response to caps-PS, which are considered thymus-independent Ags. Administration of MR1, an antagonist mAb against murine CD40L, in BALB/c mice immunized with Pneumovax resulted in an inhibition of the IgM and IgG Ab response for various caps-PS serotypes. Evidence for the involvement of CD4(+) T lymphocytes in the Ab response to caps-PS was obtained in SCID/SCID mice that, when reconstituted with B lymphocytes and CD4(+) T lymphocytes, mounted a higher specific IgM response compared with SCID/SCID mice reconstituted with only B lymphocytes. This helper effect of CD4(+) T lymphocytes was abrogated by MR1. Blocking CD40L in vitro decreased the IgM response to caps-PS and abolished the helper effect of CD4(+) T lymphocytes. CD8(+) T lymphocyte-depleted murine spleen cells mounted a higher in vivo immune response than total murine spleen cells, which provided evidence for a suppressive role of CD8(+) T lymphocytes on the anti-caps-PS immune response. CD4(+) T lymphocyte-depleted murine spleen cells, leaving a B and CD8(+) T lymphocyte fraction, elicited only a weak in vivo and in vitro Ab response, which was enhanced after MR1 administration. In summary, our data provide evidence that T lymphocytes contribute to the regulation of the anti-caps-PS immune response in a CD40L-dependent manner.     

Effector functions of CD8-positive guinea pig T lymphocytes

The response of guinea pig T lymphocytes to different stimuli was analysed with focus on the functions of CD8-positive T cells, which so far had been poorly defined in this animal model. For identification and purification of guinea pig cytotoxic T lymphocytes, three monoclonal antibodies, directed against the CD8 differentiation antigen were characterized and compared with respect to expression pattern and biochemical characteristics of the corresponding cell surface antigen. The antibodies were used for the identification of the cytotoxic T lymphocyte subpopulation within alloreactive T cell lines, and for the depletion of CD8-positive cells in in vitro assays. Purified CD4- and CD8-positive cells were tested for their ability to proliferate in response to antigen, mitogen or anti-guinea pig Thy-1 monoclonal antibodies. Both, CD4- and CD8-positive cells showed IL-2 release and subsequent proliferation after polyclonal stimulation. Cytotoxic activity in CD8-positive alloreactive T cells was expressed in vitro only after repeated stimulation.