Functional expression of the chemokine receptor CCR5 on virus epitope-specific memory and effector CD8+ T cells

Because the chemokine receptor CCR5 is expressed on Th1 CD4(+) cells, it is important to investigate the expression and function of this receptor on other T cells involved in Th1 immune responses, such as Ag-specific CD8(+) T cells, which to date have been only partially characterized. Therefore, we analyzed the expression and function of CCR5 on virus-specific CD8+ T cells identified by HLA class I tetramers. Multicolor flow cytometry analysis demonstrated that CCR5 is expressed on memory (CD28+CD45RA-) and effector (CD28-CD45RA- and CD28-CD45RA+) CD8+ T cells but not on naive (CD28+CD45RA+) CD8+ T cells. CCR5 expression was much lower on two effector CD8+ T cells than on memory CD8+ T cells. Analysis of CCR7 and CCR5 expression on the different types of CD8+ T cells showed that memory CD8+ T cells have three phenotypic subsets, CCR5+CCR7-, CCR5+CCR7+, and CCR5-CCR7+, while naive and effector CD8+ T cells have CCR5-CCR7+ and CCR5+CCR7- phenotypes, respectively. These results suggest the following sequence for differentiation of memory CD8+ T cells: CCR5-CCR7+-->CCR5+CCR7+-->CCR5+CCR7-. CCR5+CD8+ T cells effectively migrated in response to RANTES, suggesting that CCR5 plays a critical role in the migration of Ag-specific effector and differentiated memory CD8+ T cells to inflammatory tissues and secondary lymphoid tissues. This is in contrast to CCR7, which functions as a homing receptor in migration of naive and memory CD8+ T cells to secondary lymphoid tissues.     

Differential CMV-specific CD8+ effector T cell responses in the lung allograft predominate over the blood during human primary infection

Acquisition of T cell responses during primary CMV infection in lung transplant recipients (LTRs) appear critical for host defense and allograft durability, with increased mortality in donor+/recipient- (D+R-) individuals. In 15 D+R- LTRs studied, acute primary CMV infection was characterized by viremia in the presence or absence of pneumonitis, with viral loads higher in the lung airways/allograft compared with the blood. A striking influx of CD8+ T cells into the lung airways/allograft was observed, with inversion of the CD4+:CD8+ T cell ratio. De novo CMV-specific CD8+ effector frequencies in response to pooled peptides of pp65 were strikingly higher in lung mononuclear cells compared with the PBMC and predominated over IE1-specific responses and CD4+ effector responses in both compartments. The frequencies of pp65-specific cytokine responses were significantly higher in lung mononuclear cells compared with PBMC and demonstrated marked contraction with long-term persistence of effector memory CD8+ T cells in the lung airways following primary infection. CMV-tetramer+CD8+ T cells from PBMC were CD45RA- during viremia and transitioned to CD45RA+ following resolution. In contrast, CMV-specific CD8+ effectors in the lung airways/allograft maintained a CD45RA- phenotype during transition from acute into chronic infection. Together, these data reveal differential CMV-specific CD8+ effector frequencies, immunodominance, and polyfunctional cytokine responses predominating in the lung airways/allograft compared with the blood during acute primary infection. Moreover, we show intercompartmental phenotypic differences in CMV-specific memory responses during the transition to chronic infection.     

Human virus-specific CD8+ CTL clones revert from CD45ROhigh to CD45RAhigh in vivo: CD45RAhighCD8+ T cells comprise both naive and memory cells.

It has been generally believed that human CD8+ memory cells are principally found within the CD45ROhigh population. There are high frequencies of CD8+ memory CTL specific for the human CMV tegument phosphoprotein pp65 in PBMC of long-term virus carriers; the large population of memory CTL specific for a given pp65 peptide contains individual CTL clones that have greatly expanded. In this study, we found high frequencies of pp65 peptide-specific memory CTL precursors in the CD45ROhighCD45RA- population, but also appreciable frequencies in the CD45RAhigh subpopulation. Because the majority of CD8+ T cells in PBMC are CD45RAhigh, more of the total pp65-specific memory CTL pool is within the CD45RAhigh than in the CD45ROhigh compartment. Using clonotypic oligonucleotide probes to quantify the size of individual pp65-specific CTL clones in vivo, we found the CD45RAhigh population contributed 6- to 10-fold more than the CD45ROhigh population to the total virus-specific clone size in CD8+ cells. During primary CMV infection, an individual virus-specific CTL clone was initially CD45ROhigh, but after resolution of infection this clone was detected in both the CD45ROhigh and the CD45RAhigh populations. We conclude that CD45RA+ human CD8+ T cells do not solely comprise naive cells, but contain a very significant proportion of memory cells, which can revert from the CD45ROhigh to CD45RAhigh phenotype in vivo.     

EBV-specific CD8+ T cell memory: relationships between epitope specificity, cell phenotype, and immediate effector function

EBV infection in humans induces CD8+ T cell memory to viral epitopes derived from both lytic and latent cycle Ags. We have analyzed the relationship between the phenotype and function of the memory pool of T cells specific for these Ags. Lytic epitope-specific populations were heterogeneous in terms of CD45RO/RA and CD28 expression, whereas latent epitope-specific populations were uniformly CD45RO+ and CD28+, consistent with the higher antigenic challenge from lytic epitopes driving some memory cells toward a CD45RA+, CD28- phenotype. However, both types of memory population showed immediate epitope-specific cytotoxicity and type 1 cytokine production in ex vivo assays. Cytotoxic function was not associated with preactivated T cells, as EBV-specific populations were negative for activation markers such as CD69 or CD38, nor could cytotoxic function be ascribed to CD27- or CD56+ subsets, as such cells were not detected in EBV-specific memory. Furthermore, cytotoxicity was not limited to CD45RA+ and/or CD28- fractions, but also was observed in CD45RO+, CD28+ populations in lytic and latent epitope-specific memory. Cytokine (IFN-gamma, TNF-alpha) responses, measured by intracytoplasmic staining after peptide stimulation, also were detectable in CD45RO+ and RA+ subsets as well as CD28+ and CD28- subsets. Of other markers that were heterogeneous in both lytic and latent epitope populations, CCR7 gave the best discrimination of functionality; thus, CCR7+ cells consistently failed to give an IFN-gamma or TNF-alpha response, whereas many CCR7- cells were responsive. Our data are consistent with effector functions having a broad distribution among phenotypically distinct subsets of "effector memory" cells that have lost the CCR7 marker.     

Decreased effector memory CD45RA+ CD62L- CD8+ T cells and increased central memory CD45RA- CD62L+ CD8+ T cells in peripheral blood of rheumatoid arthritis patients

Although a role for CD8+ T cells in the pathogenesis of rheumatoid arthritis (RA) has been suggested, the precise nature of their involvement is not fully understood. In the present study we examined the central and effector memory phenotypes of CD4+ and CD8+ T cells in the peripheral blood of patients with RA and systemic lupus erythematosus. Terminally differentiated effector memory CD45RA+CD62L-CD8+ T cells were significantly decreased in RA patients, whereas the central memory CD45RA-CD62L+ CD8+ T-cell population was increased as compared with levels in healthy control individuals. Na?ve and preterminally differentiated effector memory CD45RA-CD62L- CD8+ T cells did not differ between RA patients and control individuals. The CD45RA-CD62L+ central memory CD4+ T-cell subpopulation was increased in RA patients, whereas the na?ve and effector memory phenotype of CD4+ T cells did not differ between RA patients and control individuals. In patients with systemic lupus erythematosus the distribution of na?ve/memory CD4+ and CD8+ T cells did not differ from that in age- and sex-matched control individuals. These findings show that peripheral blood CD8+ T cells from RA patients exhibit a skewed maturation phenotype that suggests a perturbation in the homeostasis of these cells. The central memory CD45RA-CD62L+ CD4+ and CD8+ T-cell numbers were increased in RA, suggesting an accelerated maturation of na?ve T cells. The decreased numbers of terminally differentiated CD45RA+CD62L- effector memory CD8+ T cells in peripheral blood of RA patients may reflect increased apoptosis of these cells or enhanced migration of these cells to sites of inflammation, which may play a role in the pathogenesis of RA.     

Phenotypic comparison of the three populations of human lymphocytes defined by CD45RO and CD45RA expression.

Published reports indicate that CD45RO-CD45RAbright T cells are native T cells, CD45RObrightCD45RA- T cells are memory T cells, and that concomitant loss of CD45RA expression and gain of CD45RO expression occurs during transition from naive to memory status. Thus, following in vitro activation of CD45RO- CD45RAbright T cells, a subset of transitional CD45ROdimCD45RAdim T cells is observed before conversion to a CD45RObrightCD45RA- phenotype is completed. Interestingly, all three of these phenotypic subsets are represented in the circulating human lymphocyte pool. We thus used dual-color flow cytometry to phenotypically characterize CD45RObrightCD45RA-, CD45ROdimCD45RAdim, and CD45RO- CD45RAbright lymphocytes. Both the CD45RObrightCD45RA- and CD45ROdimCD45RAdim subsets consisted almost entirely of T cells, whereas the CD45RO-CD45RAbright subset contained T cells plus essentially all of the B and natural killer cells. Additional studies used three-color flow cytometry to assess activation markers on T cells within the three subsets defined by CD45RO/CD45RA expression. CD25 expression increased with conversion from naive to memory status (5% of CD45RO-CD45RAbright, 24% of CD45ROdimCD45RAdim, and 42% of CD45RObrightCD45RA- T cells), whereas CD38 expression decreased during conversion (76, 53, and 27%, respectively). We also assessed the fluorescent intensities of CD11a, CD2, and CD44, shown by others to be increased on memory, compared to naive T cells. Visual inspection of fluorescence cytograms confirmed these findings, and further showed that transitional T cells express these markers at levels indistinguishable from those for naive T cells. These findings suggest that acquisition of CD25 and loss of CD38 occur relatively early in the naive-to-memory transition process, being evident in the transitional cell subset. In contrast, increased expression of CD11a, CD2, and CD44 appear to represent late events, occurring after loss of CD45RA and gain of CD45RO has been completed.     

Ability of human T lymphocytes to adhere to vascular endothelial cells and to augment endothelial permeability to macromolecules is linked to their state of post-thymic maturation

The accumulation of mononuclear cells at sites of chronic inflammation is dependent on a number of factors including localized adherence of lymphocytes to vascular endothelial cells (EC), cytokine-mediated increased adhesiveness of endothelium, chemotactic factors and endothelial permeability. The present study investigates two of the above attributes of lymphocyte-EC interaction: namely, the ability of maturationally distinct subpopulations of human T lymphocytes to adhere to vascular EC and to increase vascular endothelial permeability to macromolecules in an in vitro model. Thus, human T lymphocytes were separated into CD4+ CD8-helper/inducer, CD4- CD8+ cytotoxic/suppressor, CD29+ CD45RA- CD45RO+ memory, and CD29- CD45RA+ CD45RO- naive/virgin T subpopulations, were activated with PHA and PMA, and then examined for their adherence to EC and also for their effect on endothelial permeability. Upon activation, cells within each of the above four subpopulations exhibited increased adherence to EC. In contrast, resting CD29+ CD45RA- CD45RO+ memory T lymphocytes exhibited two to three times greater ability to adhere to EC than their CD29- CD45RA+ CD45RO- naive/virgin counterparts. Consistent with their increased adherence to EC, CD29+ CD45RO+ memory T lymphocytes, when activated, significantly increased endothelial permeability to albumin. Although activated CD45RA+ naive T lymphocytes exhibited increased adherence to EC, these cells failed to increase significantly endothelial permeability. Similar to their polyclonal counterparts, Ag-specific CD4+ CD29+ CD45RO+ T cell clones, but not their actively released mediators, also increased endothelial permeability via a noncytolytic mechanism(s). This ability of CD29+ CD45RO+ memory T lymphocytes to augment endothelial permeability may facilitate their transendothelial migration into extravascular space. These observations may provide additional insights into molecular mechanism(s) underlying pathophysiology of localized chronic inflammatory responses in general and more specifically selective accumulation of CD29+/CD45RO+ memory T lymphocytes at sites of chronic inflammation such as rheumatoid synovium.     

Induction of cytotoxic granules in human memory CD8+ T cell subsets requires cell cycle progression

Memory CD8(+) T cell responses are thought to be more effective as a result of both a higher frequency of Ag-specific clones and more rapid execution of effector functions such as granule-mediated lysis. Murine models have indicated that memory CD8(+) T cells exhibit constitutive expression of perforin and can lyse targets directly ex vivo. However, the regulated expression of cytotoxic granules in human memory CD8(+) T cell subsets has been underexplored. Using intracellular flow cytometry, we observed that only a minor fraction of CD45RA(-)CD8(+) T cells, or of CD8(+) T cells reactive to EBV-HLA2 tetramer, expressed intracellular granzyme B (GrB). Induction of GrB-containing cytotoxic granules in both CD45RA(+) and CD45RA(-) cells was achieved by stimulation with anti-CD3/anti-CD28 mAb-coated beads, required at least 3 days, occurred after several rounds of cell division, and required cell cycle progression. The strongest GrB induction was seen in the CCR7(+) subpopulations, with poorest proliferation being observed in the CD45RA(-)CCR7(-) effector-memory pool. Our results indicate that, as with naive T cells, induction of cytotoxic granules in human Ag-experienced CD8(+) T cells requires time and cell division, arguing that the main numerical advantage of a memory T cell pool is a larger frequency of CTL precursors. The fact that granule induction can be achieved through TCR and CD28 ligation has implications for restoring lytic effector function in the context of antitumor immunity.     

Identification of naive or antigen-experienced human CD8(+) T cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8(+) T cell response

Human CMV (HCMV) infection provides an informative model of how long term human CD8(+) T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8(+) T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8(+) T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8(+) clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45RO(high) cells and the subpopulation of CD45RA(high) cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8(+) T cell memory, we found that in healthy virus carriers highly purified CD28(-)CD45RA(high)CCR7(-) cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8(+) T cells are predominantly CD28(-)CD45RO(high)CCR7(-). During convalescence, an increasing proportion of pp65-specific CD8(+) T cells were CD28(-)CD45RA(high)CCR7(-). We conclude that naive human CD8(+) T cells are CD28(+)CD45RA(high), express CCR7 but not CCR6, and are predominantly CD27(+) and L-selectin CD62 ligand-positive. The phenotype CD27(+)CD45RA(high) should not be used to identify naive human CD8(+) T cells, because CD27(+)CD45RA(high) cells also contain a significant subpopulation of CD28(-)CD27(+) Ag-experienced expanded clones. Thus CD8(+) T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45RO(high) and CD28(-)CD45RA(high) T cell pools.     

Human autoreactive CD4+ T cells from naive CD45RA+ and memory CD45RO+ subsets differ with respect to epitope specificity and functional antigen avidity

T cells with specificity for self-Ags are normally present in the peripheral blood, and, upon activation, may target tissue Ags and become involved in the pathogenesis of autoimmune processes. In multiple sclerosis, a demyelinating disease of the CNS, it is postulated that inflammatory damage is initiated by CD4+ T cells reactive to myelin Ags. To investigate the potential naive vs memory origin of circulating myelin-reactive cells, we have generated myelin basic protein (MBP)- and tetanus toxoid-specific T cell clones from CD45RA+/RO- and CD45RO+/RA- CD4+ T cell subsets from the peripheral blood of multiple sclerosis patients and controls. Our results show that 1) the response to MBP, different from that to TT, predominantly emerges from the CD45RA+ subset; 2) the reactivity to immunodominant MBP epitopes mostly resides in the CD45RA+ subset; 3) in each individual, the recognition of single MBP epitopes is skewed to either subset, with no overlap in the Ag fine specificity; and 4) in spite of a lower expression of costimulatory and adhesion molecules, CD45RA+ subset-derived clones recognize epitopes with higher functional Ag avidity. These findings point to a central role of the naive CD45RA+ T cell subset as the source for immunodominant, potentially pathogenic effector CD4+ T cell responses in humans.     

Heterogeneous mechanisms of human cytotoxic T lymphocyte generation. I. Differential helper cell requirement for the generation of cytotoxic effector cells from CD8+ precursor subpopulations.

The subpopulations of CD8+ T cells defined by CD45RA Ag expression have been hypothesized to represent cells varying in their relative maturation along a common, activation-dependent differentiation pathway. Previous studies have shown that both the CD8+CD45RA+ and CD8+CD45RA- subsets contain precursor cells capable of developing into alloreactive CTL. In the current study, we have examined the mechanisms involved in the generation of CTL effector cells from these two CD8+ subsets. Purified CD8+CD45RA+ or CD8+CD45RA- cells were stimulated with allogeneic non-T cells, either alone or in the presence of CD4+ Th cells. Although the generation of CTL from CD8+CD45RA- precursor cells consistently required the presence of CD4+ Th cells, cytotoxic effector cells could be generated from CD8+CD45RA+ precursor cells in the absence of CD4+ cells. Several lines of evidence indicated that the helper cell-independent generation of cytotoxic effector cells from CD8+CD45RA+ precursors resulted from the unique ability of this subset to produce and use IL-2 in an autocrine fashion: 1) exogenous IL-2 could replace the effects of CD4+ helper cells for either CD8+ subset; 2) the helper cell-independent functional maturation of CD8+CD45RA+ cells could be blocked by anti-CD25 or anti-IL-2 antibodies; and 3) CD8+CD45RA+ cells produced IL-2 after activation with allogeneic cells. The finding that precursors for helper cell-independent CTL generation are restricted to the CD8+CD45RA+ subset suggests that this capability may vary as a function of the maturation of CD8+ cells.     

T8 cell regulation of human B cell responsiveness: regulatory influences of CD45RA+ and CD45RA- T8 cell subsets

The immunoregulatory functions of human T8 cell subpopulations defined by mAb to the CD45RA molecule (2H4) were examined. Both CD45RA+ and CD45RA- T8 cells that had been treated with mitomycin C provided help for the production of immunoglobulins by B cells in cultures stimulated with immobilized mAb to CD3 (64.1). In contrast, both CD45RA+ and CD45RA- T8 cells that had not been treated with mitomycin C suppressed B cell responses in anti-CD3-stimulated cultures, although CD45RA+ T8 cells were more effective in this regard. Interleukin 2 (IL2) enhanced suppression by anti-CD3-activated CD45RA- T8 cells, whereas suppression by CD45RA+ T8 cells was almost maximal and not as much increased by IL2. The differentiation into suppressor-effector cells in this system appeared to involve the production of IL2, but not the production of interferon (INF)-gamma. Thus, CD45RA+ T8 cells produced higher amounts of IL2 but lower amounts of IFN-gamma than CD45RA- T8 cells in anti-CD3-stimulated cultures. Moreover, addition of mAb to the p55 component of IL2 receptor (anti-Tac) inhibited the generation of suppressor activity from CD45RA+ and CD45RA- T8 cells. The pattern and magnitude of suppression of B cell responses by CD45RA+ and CD45RA- T4 cells were similar to that by CD45RA+ and CD45RA- T8 cells in this system. Finally, preactivated CD45RA+ T8 cells that had lost CD45RA expression suppressed the B cell responses as effectively as fresh CD45RA+ T8 cells. The results indicate that both CD45RA+ and CD45RA- T8 cells can help or suppress B cell responses. More importantly, the data suggest that the suppressor-effector function of human T cells may rather be related with the stages of the post-thymic differentiation as evidenced by the expression of the CD45RA molecule than represent the fully differentiated T cell subsets, such as T4 and T8 cells. In addition, the CD45RA molecule appeared not to be involved in the suppressor-effector function, but to determine the stage of post-thymic differentiation.     

Novel immunoregulatory functions of phenotypically distinct subpopulations of CD4+ cells in the human neonate.

Although normal numbers of CD4+ T cells are present in the human neonate, cord blood CD4+ cells are deficient in their ability to provide help for antibody production. In the present studies, we have examined the cellular basis for this functional deficit by analyzing the phenotypic properties and immunoregulatory functions of the subsets of cord blood CD4+ cells defined by anti-CD45RA mAb. In contrast to CD4+ cells from adults, greater than 90% of cord blood CD4+ cells expressed the CD45RA, CD38, and Leu-8 membrane Ag. When neonatal CD4+ cells were cultured with adult B cells and PWM or anti-CD4+ mAb, no helper function was apparent. However, when the small number of CD4+CD45RA- cells in cord blood were purified and similarly analyzed, helper activity comparable to that of adult CD4+CD45RA- cells was found. This helper function was profoundly suppressed by the presence of even small numbers of cord blood (but not adult) CD4+CD45RA+ cells. Irradiation of mitomycin C treatment of neonatal CD4+CD45RA+ cells abrogated their suppressor activity, but did not induce helper capability. However, after activation with PHA and culture in IL-2, cord blood CD4+CD45RA+ cells lost their suppressor activity and acquired the ability to provide help for B cell differentiation. This functional maturation was accompanied by their conversion to the CD4+CD45RA- phenotype. Thus, whereas cord blood CD4+CD45RA+ and CD4+CD45RA- cells share certain properties with the analogous subsets in adults, our data show that the dominant immunoregulatory function of cord blood CD4+ cells is suppression mediated by CD4+CD45RA+ (and CD38+) cells. In view of these phenotypic and functional differences between neonatal and adult CD4+CD45RA+ cells, we propose that "naive" CD4+CD45RA+ cells undergo age-related maturational changes that are unrelated to their postulated activation-dependent post-thymic differentiation into CD4+CD45RA- "memory" cells capable of helper functions.     

Direct effects of interleukin-7 on the function of human T cells <Emphasis Type="Italic">in vitro</Emphasis>

CD3+ T lymphocytes were isolated by positive magnetic separation from the peripheral blood of healthy donors. In the absence of any additional activating stimuli, interleukin-7 (IL-7) was shown to augment the levels of T cells expressing CD25 activation marker both in CD4-positive and in CD4-negative effector memory (CD45RA^-CD197^-) T cell subsets, as well as in terminally differentiated (CD45RA⁺CD197^-) T cells, without significantly affecting the activation status of naive (CD45RA⁺CD197⁺) and central memory (CD45RA^-CD197⁺) T cells. In addition, IL-7 noticeably enhanced the production of IL-2, interferon-γ (IFN-γ), and IL-10, but not IL-4, in T cells. The direct effects of IL-7 on T cell activation induced in vitro by MACSiBead? particles coated with CD2, CD3, and CD28 antibodies (Abs) were also investigated. Upon cell activation, IL-7 significantly augmented the levels of CD25+ T cells in naive (CD45RA+CD197+), central memory (CD45RA^-CD197⁺), and effector memory (CD45RA^-CD197^-) T-cell compartments. In addition, IL-7 facilitated activation of CD4^- (but not CD4⁺) terminally differentiated effector (CD45RA⁺CD197^-) T cells. Finally, IL-7 was found to upregulate the production of IL-2, IFN-γ, IL-4, and IL-10 by activated T cells. In conclusion, we speculate that IL-7 is capable of enhancing functional T cell activity without causing significant functional inbalance between various T cell subsets.     

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T(EMRA) cells in early infection are linked to control of HIV-1 viremia and predict the subsequent viral load set point

CD8(+) T cells are believed to play an important role in the control of human immunodeficiency virus type 1 (HIV-1) infection. However, despite intensive efforts, it has not been possible to consistently link the overall magnitude of the CD8(+) T-cell response with control of HIV-1. Here, we have investigated the association of different CD8(+) memory T-cell subsets responding to HIV-1 in early infection with future control of HIV-1 viremia. Our results demonstrate that both a larger proportion and an absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(+) effector memory T cells (T(EMRA) cells) were associated with a lower future viral load set point. In contrast, a larger absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(-) effector memory T cells (T(EM)) was not related to the viral load set point. Overall, the findings suggest that CD8(+) T(EMRA) cells have superior antiviral activity and indicate that both qualitative and quantitative aspects of the CD8(+) T-cell response need to be considered when defining the characteristics of protective immunity to HIV-1.     

A Dichotomy in Cortical Actin and Chemotactic Actin Activity between Human Memory and Naive T Cells Contributes to Their Differential Susceptibility to HIV-1 Infection

Human memory and naive CD4 T cells can mainly be identified by the reciprocal expression of the CD45RO or CD45RA isoforms. In HIV-1 infection, blood CD45RO memory CD4 T cells are preferentially infected and serve as a major viral reservoir. The molecular mechanism dictating this differential susceptibility to HIV-1 remains largely obscure. Here, we report that the different susceptibility of memory and naive T cells to HIV is not determined by restriction factors such as Apobec3G or BST2. However, we observed a phenotypic distinction between human CD45RO and CD45RA resting CD4 T cells in their cortical actin density and actin dynamics. CD45RO CD4 T cells possess a higher cortical actin density and can be distinguished as CD45RO⁺Actin^high. In contrast, CD45RA T cells are phenotypically CD45RA⁺Actin^low. In addition, the cortical actin in CD45RO memory CD4 T cells is more dynamic and can respond to low dosages of chemotactic induction by SDF-1, whereas that of naive cells cannot, despite a similar level of the chemokine receptor CXCR4 present on both cells. We further demonstrate that this difference in the cortical actin contributes to their differential susceptibility to HIV-1; resting memory but not naive T cells are highly responsive to HIV-mediated actin dynamics that promote higher levels of viral entry and early DNA synthesis in resting memory CD4 T cells. Furthermore, transient induction of actin dynamics in resting naive T cells rescues HIV latent infection following CD3/CD28 stimulation. These results suggest a key role of chemotactic actin activity in facilitating HIV-1 latent infection of these T cell subsets.     

Effect of Cytomegalovirus Co-Infection on Normalization of Selected T-Cell Subsets in Children with Perinatally Acquired HIV Infection Treated with Combination Antiretroviral Therapy

Background

We examined the effect of cytomegalovirus (CMV) co-infection and viremia on reconstitution of selected CD4+ and CD8+ T-cell subsets in perinatally HIV-infected (PHIV+) children ≥ 1-year old who participated in a partially randomized, open-label, 96-week combination antiretroviral therapy (cART)-algorithm study.

Methods

Participants were categorized as CMV-naïve, CMV-positive (CMV+) viremic, and CMV+ aviremic, based on blood, urine, or throat culture, CMV IgG and DNA polymerase chain reaction measured at baseline. At weeks 0, 12, 20 and 40, T-cell subsets including naïve (CD62L+CD45RA+; CD95-CD28+), activated (CD38+HLA-DR+) and terminally differentiated (CD62L-CD45RA+; CD95+CD28-) CD4+ and CD8+ T-cells were measured by flow cytometry.

Results

Of the 107 participants included in the analysis, 14% were CMV+ viremic; 49% CMV+ aviremic; 37% CMV-naïve. In longitudinal adjusted models, compared with CMV+ status, baseline CMV-naïve status was significantly associated with faster recovery of CD8+CD62L+CD45RA+% and CD8+CD95-CD28+% and faster decrease of CD8+CD95+CD28-%, independent of HIV VL response to treatment, cART regimen and baseline CD4%. Surprisingly, CMV status did not have a significant impact on longitudinal trends in CD8+CD38+HLA-DR+%. CMV status did not have a significant impact on any CD4+ T-cell subsets.

Conclusions

In this cohort of PHIV+ children, the normalization of naïve and terminally differentiated CD8+ T-cell subsets in response to cART was detrimentally affected by the presence of CMV co-infection. These findings may have implications for adjunctive treatment strategies targeting CMV co-infection in PHIV+ children, especially those that are now adults or reaching young adulthood and may have accelerated immunologic aging, increased opportunistic infections and aging diseases of the immune system.     

Both Memory and CD45RA+/CD62L+ Naive CD4+ T Cells Are Infected in Human Immunodeficiency Virus Type 1-Infected Individuals

Cellular activation is critical for the propagation of human immunodeficiency virus type 1 (HIV-1) infection. It has been suggested that truly naive CD4(+) T cells are resistant to productive HIV-1 infection because of their constitutive resting state. Memory and naive CD4(+) T-cell subsets from 11 HIV-1-infected individuals were isolated ex vivo by a combination of magnetic bead depletion and fluorescence-activated cell sorting techniques with stringent criteria of combined expression of CD45RA and CD62L to identify naive CD4(+) T-cell subsets. In all patients HIV-1 provirus could be detected within naive CD45RA+/CD62L+ CD4(+) T cells; in addition, replication-competent HIV-1 was isolated from these cells upon CD4(+) T-cell stimulation in tissue cultures. Memory CD4(+) T cells had a median of fourfold more replication-competent virus and a median of sixfold more provirus than naive CD4(+) T cells. Overall, there was a median of 16-fold more integrated provirus identified in memory CD4(+) T cells than in naive CD4(+) T cells within a given patient. Interestingly, there was a trend toward equalization of viral loads in memory and naive CD4(+) T-cell subsets in those patients who harbored CXCR4-using (syncytium-inducing) viruses. Within any given patient, there was no selective usage of a particular coreceptor by virus isolated from memory versus naive CD4(+) T cells. Our findings suggest that naive CD4(+) T cells may be a significant viral reservoir for HIV, particularly in those patients harboring CXCR4-using viruses.     

Differentiation of human CD8(+) T cells from a memory to memory/effector phenotype

Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA(-)CD8(+) T cells with different CD28/CD27 phenotypes showed that CD28(+)CD45RA(-)CD8(+) and CD27(+)CD45RA(-)CD8(+) T cells have characteristics of memory T cells, whereas CD28(-)CD45RA(-)CD8(+) and CD27(-)CD45RA(-)CD8(+) T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8(+) T cells into memory/effector CD8(+) T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8(+) T cells. Three subsets of CD45RA(-)CD8(+) T cells were observed in both total CD8(+) T cells and EBV- or HCMV-specific CD8(+) T cells: CD27(+)CD28(+), CD27(+)CD28(-), and CD27(-)CD28(-). A significant number of the CD27(-)CD28(+) subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8(+) T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-)-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). This was supported by the observation that the frequency of CCR5(+) cells and CCR7(+) cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27(+)CD28(+) memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5(-)CCR7(+), CCR5(+)CCR7(+), and CCR5(+)CCR7(-). These findings suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-) (CCR5(-)CCR7(+)-->CCR5(+)CCR7(+)-->CCR5(+)CCR7(-))-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). The presence of a CD27(-)CD28(+) subset with a CCR5(+)CCR7(-) phenotype implies a specialized role for this subset in the differentiation of CD8(+) T cells.     

Functional T cell responses to tumor antigens in breast cancer patients have a distinct phenotype and cytokine signature

The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8(+) T cells, and a lower proportion of IFN-gamma-producing CD4(+) and/or CD8(+) T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8(+) T cells in breast cancer patients was almost completely CD28(+)CD45RA(-) (memory phenotype). CMV-responsive CD8(+) T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27(-)CD28(-)CD45RA(+)) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.