Naive CD4 T-cell Research Articles

Regulating Viral Myocarditis: Allografted Regulatory T Cells Decrease Immune Infiltration and Viral Load

Coxsackievirus was first discovered as a filterable agent associated with a paralytic syndrome, so named for its identification in Coxsackie, New York (coxsackievirus type A).1 Coxsackievirus type B (CVB) was isolated the following year from patients with aseptic meningitis,2 and by the mid-1950s, an association with acute myocarditis in humans was becoming clear.3–6 Many other viruses have since been shown to cause myocarditis and its long-term sequelae, arrhythmias, dilated cardiomyopathy, and heart failure. By example, adenovirus, herpesviruses, and influenza can cause myocarditis in humans and models of heart failure. Because immune cell infiltration is an easily seen feature of myocarditis, it is perhaps not surprising that the inflammatory response would be posited as a major cause of tissue injury during viral myocarditis. However, the virus itself has lytic and destructive potential, and a range of data indicate both viral and immune contributions to cardiomyocyte and interstitial damage during myocarditis. The question is often posed: Is damage primarily virus-mediated or more so the product of an overzealous immune response and autoimmunity? Perhaps the greatest error in the discussion has been approaching these 2 facets of the disease process in a mutually exclusive manner. Shi et al7 now show in this issue of Circulation that virus replication and the immune response are exquisitely intertwined in myocarditis pathogenesis. Article see p 2624 Over the last 3 decades, it has become apparent that viral load is a primary determinant of viral myocarditis severity,8–11 the virus causing extensive, early, direct injury to the myocardium through its replication. Results cited here all indicate significant injury to the myocardium before cardiac immune infiltration, supporting the view that considerable damage to the heart is directly mediated by the virus alone. An argument has been made previously for the importance of chronic virus replication …

Human Langerhans Cells Are More Efficient Than CD14−CD1c+ Dermal Dendritic Cells at Priming Naive CD4+ T Cells

Few data are available regarding the role of human skin dendritic cells (DCs) in driving T-cell responses. In this study we analyzed the relative capacity of Langerhans cells (LCs) and dermal CD14(-)CD1c(+) DCs (DDCs) to trigger naive CD4(+) T-cell proliferation and differentiation. DC subsets were purified after a 2-day migration from epidermis and dermis of the same skin sample. Migratory LCs showed far more activated phenotype than CD1c(+)DDCs and distinct expression of new molecules of the B7 family; when compared with LCs, CD1c(+)DDCs showed higher PD-L1 and lower inducible co-stimulator ligand (ICOS-L) expression. As expected, CD1c(+)DDCs showed lower allostimulatory property than LCs, a process that was partly reversed by anti-PD-L1 mAb. LCs were significantly more efficient than CD1c(+)DDCs at inducing allogeneic naive CD4(+) T cells to secrete both T helper cell 1 (Th1; IFN-gamma and tumor necrosis factor-alpha ) and Th2 (IL-4 and IL-5) cytokines. Moreover, anti-PD-L1 mAb increased the production of IFN-gamma by both LC- and CD1c(+)DDC-stimulated T cells. Globally, these results argue for a preponderant role of human LCs in inducing naive CD4(+) T-cell priming. Low expression of co-stimulatory molecules together with high expression of PD-L1 might limit the efficiency of CD1c(+)DDCs at inducing naive CD4(+) T-cell proliferation and secretion of cytokines.

CD4 T-cell hyperactivation and susceptibility to cell death determine poor CD4 T-cell recovery during suppressive HAART

The failure to increase CD4 T-cell counts in some HAART-treated HIV-infected patients with satisfactory virological responses has been related to low CD4 T-cell production, high turnover and death. However, the relative contribution of these factors is still unclear, strongly limiting the definition of appropriate therapeutic strategies for these patients. A cross-sectional study was designed to evaluate the contribution of thymic activity, microbial translocation, cellular activation and death to CD4 T-cell recovery. We included 230 HIV-infected individuals on suppressive HAART (>2 years); 95 of them were considered 'discordant' (CD4 T-cell count <350 cells/mul) and 135 were considered 'concordant'. Comparative and logistic regression analyses were performed. Discordant patients showed higher levels of activated [human leukocyte antigen (HLA)-DRCD95 and CD38CD45RA] cells in both the CD8 and CD4 T-cell compartments. Notably, the most significant differences were observed in CD4 T cells. Discordant patients showed lower naive CD4 T-cell production (CD45RACD31 cells), higher spontaneous ex-vivo CD4 T-cell death and higher plasma levels of soluble CD14. Multivariate analysis showed that activation and death of CD4 T cells, along with nadir CD4 T-cell counts, were the only predictive factors for poor immune recovery. Moreover, the low correlations found between CD4 T-cell activation or death with thymic output and bacterial translocation suggest that additional factors modulate cellular activation and death and, in turn, CD4 T-cell recovery. CD4 T-cell repopulation during HAART is determined by CD4 T-cell activation and death. Therefore, strategies aimed to reduce these parameters should be envisaged to treat discordant patients.

Differential requirements of MHC and of DCs for endogenous proliferation of different T-cell subsets in vivo

T cells transferred into severe lymphopenic hosts undergo rapid proliferation known as "endogenous proliferation" that are distinct from conventional homeostatic proliferation. Unlike homeostatic proliferation, cytokines, such as IL-7 are dispensable, yet TCR:MHC interaction is essential for this process to occur. However, cell types inducing the proliferation have not formally been addressed. In this study, we report that CD11c+ conventional DCs play irreplaceable roles in inducing endogenous proliferation of both naive and memory phenotype CD4 T cells via TCR-MHC II interaction. By contrast, CD8 T-cell endogenous proliferation was independent of MHC I or CD11c+ DCs. Interestingly, MHC II was necessary to support naive CD8 T-cell proliferation within MHC I-deficient hosts. Depletion of both B cells and DCs was sufficient to abrogate the proliferation of naive but not of memory CD8 T cells. These results suggest that depending on the T-cell lineages, as well as the differentiation status, different mechanisms control endogenous proliferation, revealing in vivo complexity of T-cell proliferation under lymphopenic conditions.

Comparison of chlordecone and estradiol effects on splenic T-cells in (NZB × NZW)F 1 mice

Previous studies have shown that treatment of ovariectomized females with 17-β estradiol (E2) accelerates the development of autoimmunity in the (NZB × NZW)F 1 murine lupus model. Treatment with estrogenic organochlorine pesticides (OCPs) such as chlordecone produces a similar effect. Although it is reasonable to postulate that the effects of chlordecone and related OCPs on autoimmunity are due to their estrogenic effects, this has not been clearly demonstrated. The objective of this study was to compare effects of chlordecone and E2 on splenic T lymphocyte parameters plausibly related to autoimmunity; specifically, on T-cell phenotype and functions. Ovariectomized (NZB × NZW)F 1 mice were treated for 6 weeks with implanted sustained-release pellets containing chlordecone or E2 at dosing rates shown previously to significantly shorten time to onset of disease. E2, but not chlordecone, increased the percentage of activated and memory CD4 T-cells, and reduced naive CD4 T-cells. E2 also elevated CD25 and glucocorticoid-induced TNF receptor (GITR) levels in CD4 T-cells, an effect not shared by chlordecone. On the other hand, both chlordecone and E2 increased Bcl-2 expression in CD4 T-cells and reduced CD4 T-cell apoptosis without affecting their proliferation. Although both treatments increased TNF-α and IL-2 secretion by CD4 T-cells, only chlordecone increased secretion of IFN-γ and GM-CSF. E2, but not chlordecone, increased IL-10 secretion. These observations indicate that although it is considered an estrogenic OCP, chlordecone exerts effects on splenic T-cells that are different in a number of ways from E2.

Short-Term Ionomycin Exposure Activates Naive Murine T-Cells and Induces a Rapid Phenotypic Shift to Memory T-Cell Status: Potential for Use as a Method To Reduce GvHD Activity of Allogeneic T-Cells.

We are investigating methods to reduce the graft-versus-host disease (GVHD) potential of donor T-cells while retaining graft-versus-leukemia (GVL) activity in allogeneic HSCT. Previous investigations by our group and others in have shown that naive CD4 T-cells induce severe acute GVHD, while memory CD4 T-cells do not induce GVHD but retain GVL activity in murine transplant models. These findings have led to studies for the development of methods to increase the number of memory T-cells available for transplant. The calcium ionophore, ionomycin, is a T-cell activating agent and mitogen. By increasing intracellular Ca2+ levels, ionomycin is induces T-cell activation through signaling mechanisms including phospholipase C activation, hydrolysis of phosphoinositides, and activation of protein kinase C. Differences in memory and naive T-cell responses to ionomycin have been attributed to resistance of memory T-cells to increases in Ca2+. Memory T-cells lack intracellular Ca2+ stores, and are also resistant to influx of Ca2+. Brief low dose ionomycin exposure (20min, 2μM) of T-cells, leading to increased density of naive T-cells, has previously been exploited as a method for separating memory and naive T-cells by Percoll gradient separation. Since ionomycin exposure induces T-cell activation through native Ca2+ dependent signaling mechanisms, we hypothesized that ionomycin-treated T-cells would shift to an activated/memory T-cell phenotype. Murine splenic T-cells were treated with 1.3μM ionomycin for 4hr. Memory and naive T-cell subsets and activation markers were analyzed by flow cytometry. 75% and 85% of untreated CD4 and CD8 T-cells, respectively, had the CD62L+ naive phenotype. These numbers were dramatically reduced to 7% and 17% after ionomycin exposure, representing a shift to the memory T-cell phenotype. Viability of T-cells was not significantly affected. The majority of remaining CD62L+ naive T-cells expressed activation markers CD25 and CD69. The fraction of CD4+CD25+Foxp3+ regulatory T-cells was also determined by intracellular staining of the transcription factor and co-expression of surface markers. CD4+CD25+Foxp3+ regulatory T-cells represented 4% of untreated CD4 T-cells and 3% of ionomycin-treated CD4 T-cells. While ionomycin has been used for many years in studies of T-cell activation, to our knowledge this is the first demonstration of a rapidly-induced shift of naive T-cells to a memory phenotype. A pilot experiment was conducted testing the GVHD activity of ionomycin-treated splenocytes (SP) in B6→ (B6 × Balb/C)CB6F1 recipients. 5 × 106 T-cell depleted bone marrow cells (TCD-BM) were transplanted along with 10 × 106 treated or untreated SP. Mice that received untreated SP all died from acute GvHD by 34 days after transplant, while all recipients of ionomycin-treated SP survived until the experiement was terminated at day 49 (average weight loss was 25%, data not shown). Continuing experients will refine the dose to further reduce GVHD symptoms and also test GVL activity of the treated cells. Treatment of donor T-cells with ionomycin may represent a clinically applicaple method to engineer donor lymphocyte infusions that are safer for HSCT patients. [Display omitted]

Dose‐dependent modulation of CD8 and functional avidity as a result of peptide encounter

The generation of an optimal CD8(+) cytotoxic T lymphocyte (CTL) response is critical for the clearance of many intracellular pathogens. Previous studies suggest that one contributor to an optimal immune response is the presence of CD8(+) cells exhibiting high functional avidity. In this regard, CD8 expression has been shown to contribute to peptide sensitivity. Here, we investigated the ability of naive splenocytes to modulate CD8 expression according to the concentration of stimulatory peptide antigen. Our results showed that the level of CD8 expressed was inversely correlated with the amount of peptide used for the primary stimulation, with higher concentrations of antigen resulting in lower expression of both CD8alpha and CD8beta. Importantly the ensuing CD8(low) and CD8(high) CTL populations were not the result of the selective outgrowth of naive CD8(+) T-cell subpopulations expressing distinct levels of CD8. Subsequent encounter with peptide antigen resulted in continued modulation of both the absolute level and the isoform of CD8 expressed and in the functional avidity of the responding cells. We propose that CD8 cell surface expression is not a static property, but can be modulated to 'fine tune' the sensitivity of responding CTL to a defined concentration of antigen.

A Pilot Study Evaluating Time to CD4 T-cell Count &lt;350 cells/mm3 After Treatment Interruption Following Antiretroviral Therapy ± Interleukin 2: Results of ACTG A5102

Although an intermittent antiviral treatment (ART) strategy may limit long-term toxicity and cost, there is concern about the risk for virologic failure, selection of drug resistance mutations, and disease progression. By boosting CD4 T-cell counts, interleukin 2 (IL-2) could safely prolong the duration of treatment interruption (TI) in a CD4-driven strategy. The AIDS Clinical Trials Group (ACTG) study A5102 evaluated 3 cycles of IL-2 before TI, on clinical and immunologic outcomes, using a CD4 T-cell count of <350 cells/mm as the threshold for restarting ART. Forty-seven HIV-infected subjects on potent ART with CD4 T-cell counts of > or =500 cells/mm or more and HIV RNA levels of less than 200 copies/mL were randomized to arm A (ART + three 5-day cycles of IL-2 at 4.5 million U, Sc, BID every 8 weeks, n = 23) or arm B (ART alone, n = 24) for 18 weeks (step 1). At the end of step 1, subjects with a CD4 T-cell count of > or =500 cells/mm or more stopped ART until a CD4 count of <350 cells/mm (step 2). CD4 T-cell count, time to return of viremia, and the emergence of drug resistance mutations after TI were compared between study arms. IL-2 recipients maintained higher CD4 counts during TI for 48 weeks with a waning of the CD4 effect by 72 weeks. A sustained CD4 T-cell count of more than 350 cells/mm and more durable TI were associated with a higher nadir CD4 T-cell count before ART and higher naive CD4 T-cell count at entry. After TI, a higher viral set point and drug resistance mutations at virologic rebound were associated with a shorter time to CD4 T-cell count of less than 350 cell/mm. There were no differences in the magnitude of virologic rebound (at week 8 of step 2, median log10 HIV RNA level was 4.23 for arm A and 4.21 for arm B) or the steady-state HIV-1 RNA level after week 8. IL-2 before TI did not prolong time to CD4 of less than 350 cells/mm. A TI strategy utilizing a CD4 T-cell threshold of less than 350 cells/mm for restarting ART appears generally safe with most subjects in both arms remaining off ART for more than 1 year. Implications of our results for TI strategies include the potential advantage of starting ART at higher CD4 T-cell levels while avoiding any drug resistance and evaluating immunomodulators or drugs to reduce T-cell activation and HIV-1 RNA rebound during the TI.

Interruption of antiretroviral therapy blunts but does not abrogate CD4 T-cell responses to interleukin-2 administration in HIV infected patients

Intermittent administration of interleukin (IL)-2 to HIV infected patients leads to CD4 T-cell expansions that are associated with decreased CD4 T-cell turnover. IL-2 is under evaluation in antiretroviral therapy (ART) interruption studies, but it is unclear how the emergence of viremia may affect CD4 expansions. CD4 T-cell responses were evaluated in 27 HIV infected patients on long-term intermittent IL-2 therapy who underwent ART interruption immediately after an IL-2 cycle ('IL-2/off') and compared with responses from a previous IL-2 cycle while on continuous ART ('IL-2/on'). Immunophenotypic analysis, including intracellular Ki67 staining, of cryopreserved peripheral blood mononuclear cells was performed. CD4 T-cell increases, in naive and central memory CD4 T-cell subsets, were observed in the IL-2/on (106 and 327 cells/microl, respectively) and IL-2/off (84 and 184 cells/microl, respectively) cycles 1 month following IL-2 administration. These increases were greater during the IL-2/on cycle (P = 0.05, P = 0.01, respectively). In both cycles, the change in CD4 T-cell count correlated with the change in CD4/CD25 T cells. In the IL-2/off cycle, the change in the proportion of CD4 T cells expressing Ki67 was associated with both the changes in viral load (r = 0.64, P = 0.001) and the changes in CD4 T cells (r = -0.56, P = 0.01). IL-2 administration followed by ART interruption led to significant, although blunted, CD4 T-cell increases. IL-2 induced CD4 T-cell increases in the setting of emergent viremia were associated with decreased CD4 T-cell activation that counteracted the viremia-induced increases in CD4 T-cell activation.

Immunologic Recovery after Nonmyeloablative (NM) Conditioning: A Comparison between Related Versus Unrelated Donor Hematopoietic Cell Transplantation (HCT).

Background. After NM conditioning, recipients of peripheral blood stem cells (PBSC) from HLA-identical siblings (MRD) have persistent recipient-derived immunity leading to lower incidence of early infectious complications in comparison with patients (pts) given myeloablative conditioning (Exp Hematol 31: 941, 2003). Because of greater genetic disparities, intensity and duration of postgrafting immunosuppression (IS) have been increased in pts given PBSC from HLA-matched unrelated donors (URD), in order to both promote engraftment and decrease risk of GVHD. This study was done to determine how this increased/extended postgrafting IS affected immune recovery and infectious complications.Methods. We compared immunologic recovery in 51 pts given PBSC from MRD (n=51) and 43 pts given grafts from URD after 2 Gy TBI +/− fludarabine. Postgrafting IS concisted of mycophenolate mofetil (MMF, given TID for 40 days followed by a 56 day taper in URD recipients, and BID for 28 days in MRD recipients) and cyclosporin. The counts of blood mononuclear cell subsets were assessed by flow cytometry; frequency of CMV-specific CD4 T-helper cells among CMV seropositive pts or CMV-seronegative pts with CMV-seropositive donors was determined by lymphoproliferation (LPR), limiting-dilution assays (LDA), and intracellular interferon-gamma (IF) production (ICC). Analyses were performed on days 30, 80, 180 and 365 after HCT.Results. On day 30 after HCT, URD recipients had lower counts of total B-cells (P=.02), naive B-cells (P=.03), memory B-cells (P=.01), CD4 T-cells (P=.06), naive CD4 T-cells (P=.05), memory CD4 T-cells (P=.003), CD8 T-cells (P=.0004), naive CD8 T-cells (P=.08) and memory CD8 T-cells (P=.006) than MRD recipients. However, the counts of mononuclear cell subsets were similar in URD and MRD recipients from day 30 to day 365 after HCT. Similarly, the frequency of CMV-specific T-helper cells was significantly lower in URD than in MRD recipients on day 30 after HCT as determined by LPR (median CPM 17 versus 92 per 1000 CD4 T-cells, P=.02), LDA (median 22307 versus 73251 CMV-CD4 T-helper cells per L, P=.7) and ICC (median 0.26% versus 0.93% CD4 cells positive for IF, P=.02). This delay in CMV-specific immune reconstitution translated into increased frequency of CMV-reactivation (and increased use of preemptive antiviral therapy [PET]) among CMV-seropositive pts or CMV-seronegative pts with CMV seropositive donors given URD grafts (63%) compared with MRD (33%) recipients (P=.02) the first 100 days after HCT. This did not lead to increased CMV disease among URD recipients (1 episode) compared with MRD recipients (1 episode), demonstrating that PET was similarly effective in preventing CMV diseases in both groups. After day 100, there were no statistically significant differences in immune reconstitution parameters. Incidence figures for non-CMV viral infections, bacteremias, and invasive fungal infections were not significantly different between the 2 groups. A multivariate analysis assessing pre-transplant and post-transplant factors associated with immunologic recovery after NM conditioning will be presented.Conclusions. Despite similar NM conditioning regimens, immunologic recovery was delayed among URD recipients in comparison to MRD recipients, probably because of increased/extended postgrafting IS. This led to increased incidence of CMV reactivation among URD recipients. CMV infection and immune reconstitution pattern appear similar to those seen after myeloablative PBSC transplantation.

Ex Vivo Fludarabine Selectively Depletes Human Naive T-Cell Subsets: A Step Toward Modifying Donor Lymphocyte Infusion.

Ex vivo modification of donor lymphocytes with purine analogs (mDL) may help to minimize graft versus host disease (GvHD) while providing beneficial graft versus leukemia (GvL) effects. In a murine model system, we have shown that allogeneic donor splenocytes, treated with fludarabine ex vivo have significantly reduced GvHD activity when transferred to irradiated recipient mice, and retain anti-viral and GvL activities (Giver, 2003). This effect appears to be mediated by relative depletion of donor CD4 CD44low, “naive” T-cells. As a first step toward developing mDL for use in patients, we sought to evaluate the effects of ex vivo fludarabine exposure on human T-cell subsets, and to determine the minimum dose of fludarabine required to achieve this effect.Methods: Peripheral blood mononuclear cell samples from 6 healthy volunteers were evaluated at 0, 24, 48, and 72 hour time points after ex vivo incubation in varying dosages of fludarabine: 2, 5, and 10(n=3) mcg/ml. Fludarabine incubated samples were compared to samples that received no fludarabine (untreated). The total viable cell number was determined and the fractions and absolute numbers of viable CD4 and CD8 naïve and memory T-cells were determined using flow cytometry after incubation with 7-AAD (dead cell stain), CD4, CD8, CD45RA, CD62L, and CCR7 antibodies, and measuring the total viable cells/ml.Results: The numbers of viable CD4 and CD8 T-cells remained relatively stable in control cultures. Without fludarabine, the average viability at 72 hr of naive and memory T-cells were 92% and 77% for CD4 and 86% and 63% for CD 8 (Fig. 1A). Naive CD4 T-cells were more sensitive to fludarabine-induced death than memory CD4 cells. At 72 hr, the average viability of fludarabine-treated naive CD4 T-cells was 33% at 2 mcg/ml (8.2X the reduction observed in untreated cells) and 30% at 5 mcg/ml, while memory CD4 T-cells averaged 47% viability at 2 mcg/ml (2.3X the reduction observed in untreated cells) (Fig. 1B) and 38% at 5 mcg/ml. The average viability of naive CD8 T-cells at 72 hr was 27% at 2 mcg/ml and 20% at 5 mcg/ml, while memory CD8 T-cell viability was 22% at 2 mcg/ml and 17% at 5 mcg/ml. Analyses on central memory, effector memory, and Temra T-cells, and B-cell and dendritic cell subsets are ongoing. The 5 and 10 mcg/ml doses also yielded similar results in 3 initial subjects, suggesting that 2 mcg/ml or a lower dose of fludarabine is sufficient to achieve relative depletion of the naive T-cell subset.Conclusions: Future work will determine the minimal dose of fludarabine to achieve this effect, test the feasibility of using ex vivo nucleoside analog incubation to reduce alloreactivity in samples from patient/donor pairs, and determine the maximum tolerated dose of mDL in a phase 1 clinical trial with patients at high risk for relapse and infectious complications following allogeneic transplantation. [Display omitted]

Human Naive CD4 T-Cell Clones Specific for HIV Envelope Persist for Years In Vivo in the Absence of Antigenic Challenge

To study the persistence of HIV-specific human naive CD4-lymphocytes in vivo in the absence of antigenic stimulation, we identified 2 HIV-seronegative low-risk subjects carrying CD4-cells specific for gp120 that could be expanded in vitro. CD4 T-cell lines specific for gp120 were generated by stimulation cycles with antigen-pulsed antigen-presenting cells. Clonal analysis was performed by spectratyping and by sequencing of the CDR3 regions of the BV and AV-T-cell receptor (TCR) genes. HIV-specific T cells were expanded in vitro in 1989 and 2004. These lines were generated from naive precursors. Analysis of TCR-BV gene family use and sequencing of the TCR-BV22 hypervariable region revealed a BV22 clonotype in the 1989 line. The BV22-CDR3-based polymerase chain reaction primer confirmed that the 1989 and 2004 T-cell lines contained the same clonotype. In addition, the 1989 and 2004 T cells used the same TCR-AV38 gene family and identical CDR3-AV regions, confirming clonal identity. Similar data for a persistent clonotype defined by BV CDR3 sequencing were obtained from the second subject. In conclusion, naive CD4-cells specific for an HIV antigen not encountered in vivo persisted for more than 10 to 15 years. An extended lifespan, homeostatic proliferation, or the ability of the thymus to issue the same CD4 T-cell clone reiteratively might account for the phenomenon.

Molecular mechanisms of apoptosis in the cells of the immune system in human aging

Aging is associated with progressive decline in immune functions and increased frequency of infections, autoimmunity, and cancer. Among immune functions, a decline in T-cell functions during aging predominates. In this review, I discuss the molecular signaling of three distinct pathways of apoptosis, namely the death receptor pathway, the mitochondrial pathway, and the most recently described endoplasmic reticulum stress pathway, and the relative sensitivity of naive, central memory, and effector memory CD8(+) T-cell subsets to apoptosis. In addition, I review apoptosis, especially via death receptor pathway, in naive and various memory subsets of CD4(+) and CD8(+) T cells (with primary emphasis on CD8(+) naive and memory subsets) in human aging and discuss the role of apoptosis in immune senescence.

Accelerated naive CD8 T-cell homing during acute cellular rejection after clinical heart transplantation

Accelerated naive CD8 T-cell homing during acute cellular rejection after clinical heart transplantation

Synthesis of several chemokines but few cytokines by primed uncommitted precursor CD4 T cells suggests that these cells recruit other immune cells without exerting direct effector functions.

Antigen-stimulated naive CD4 T cells may differentiate into effector T cells such as Th1 and Th2 cells, or may remain as proliferating but uncommitted, primed, precursor cells (Thpp cells) that can subsequently differentiate into Th1 or Th2 cells in appropriate cytokine environments. To examine potential Thpp effector functions, we compared the genes expressed by mouse Thpp, naive, Th1 and Th2 cells, using Affymetrix GeneChip and RNase Protection assays. Similar to naive CD4 T cells, Thpp cells expressed IL-2 but not the cytokines characteristic of differentiated Th1 or Th2 cells, such as IFN-gamma, IL-4, or IL-5. However, Thpp, Th1 and Th2 cells, but not naive cells, expressed several CC chemokines including CCL1/TCA3, CCL5/RANTES, CCL3/MIP-1 alpha, CCL4/MIP-1 beta, and CCL9/MIP-1 gamma. Secretion of the corresponding proteins was confirmed by ELISA and Elispot. Consistent with this chemokine expression, supernatants of activated Thpp, Th1 and Th2 cells but not naive CD4T cells induced pertussis toxin-sensitive chemotaxis of B and T cells. Supernatants of Thpp cells did not bias differentiation of naive CD4 T cells towards either Th1 or Th2 cells. The secretion of several chemokines, but few cytokines, by primed uncommitted Thpp cells suggests that their activation during an immune response may recruit effector cells without directly polarizing effector functions.

Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3ζ expression

Human effector T cells have been difficult to isolate and characterize due to their phenotypic and functional similarity to the memory subset. In this study, a biochemical approach was used to analyze human effector CD4 T cells generated in vitro by activation with anti-CD3 and autologous monocytes for 3 to 5 days. The resultant effector cells expressed the appropriate activation/differentiation markers and secreted high levels of interferon gamma (IFN-gamma) when restimulated. Biochemically, effector CD4 T cells exhibited increases in total intracellular tyrosine phosphorylation and effector-associated phosphorylated species. Paradoxically, these alterations in tyrosine phosphorylation were concomitant with greatly reduced expression of CD3zeta and CD3epsilon signaling subunits coincident with a reduction in surface T-cell receptor (TCR) expression. Because loss of CD3zeta has also been detected in T cells isolated ex vivo from individuals with cancer, chronic viral infection, and autoimmune diseases, the requirements and kinetics of CD3zeta down-regulation were examined. The loss of CD3zeta expression persisted throughout the course of effector T-cell differentiation, was reversible on removal from the activating stimulus, and was modulated by activation conditions. These biochemical changes occurred in effector T cells generated from naive or memory CD4 T-cell precursors and distinguished effector from memory T cells. The results suggest that human effector T-cell differentiation is accompanied by alterations in the TCR signal transduction and that loss of CD3zeta expression may be a feature of chronic T-cell activation and effector generation in vivo. (Blood. 2001;97:3851-3859)

Retroviral Antibody Binding of the MHC Class II Molecule: A Biochemical Influence on CD4 T Cell Differentiation in HIV Infection?

Retroviral antibody capable of binding to the major histocompatibility complex (MHC) Class II molecule has been documented in human immunodeficiency virus-1 (HIV-1)-infected patients. Interactions between the MHC Class II receptor and the T-cell receptor (TCR) are central to the immune response. Importantly, retroviral antibody possesses a much higher binding affinity for the MHC Class II receptor, when compared to the TCR. Experiments have manipulated a number of factors related to antigen-presenting cell (APC) interaction with differentiating T-cells. These studies have observed the effects of lowering antigen dose and reducing ligand density on precursor Th (T helper) cell differentiation. Studies have also examined the effect of downregulated MHC Class II receptors and co-stimulatory molecules on APC–Th cell interaction. In addition, the sequestration of antigens away from the Class II processing pathway has been studied. These investigations reveal a general trend that can determine whether a naive CD4 T-cell becomes a Th1 or Th2-like cell. If the experimental manipulation weakens the APC-Th cell interaction, a weak ligating TCR signal results. Consequently, a weak ligating TCR signal can influence precursor Th cells to become Th2-like cells. Retroviral antibody binding of MHC Class II receptors may mimic a number of experimental conditions responsible for creating a weak ligating TCR signal.

CD4 T-cell memory can persist in the absence of class II.

To understand how memory CD4 T cells are generated we have re-examined the requirements for continuing antigen stimulation in the generation and persistence of this population. We find that specific antigen is only required for a short period during the activation of naive CD4 T cells and is not required for memory generation from activated CD4 T cells or for persistence of resting memory cells generated by transfer of activated CD4 to adoptive hosts. Moreover, transfer of activated CD4 T cells to class-II-deficient hosts, indicates that TcR-class II major histocompatibility interaction is also unnecessary for either the transition from activated CD4 T cell to resting memory cells or for persistence over an eight-week period. Thus the signals regulating generation and maintenance of memory are fundamentally different from those which regulate the expansion of effector CD4 T-cell populations which include antigen itself and the CD4 T-cell autocrine cytokines induced by antigen.

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.

Do B cells drive the diversification of immune responses?

Although the phenomenon of immunodominance of epitopes within protein antigens is well established, immune responses in which antigen persists are characterized by a great diversity of Tand B-cell responses. This is observed especially in chronic infection and autoimmunity. In this brief paper, we would like to propose a mechanism for diversification of immune responses based on the unique ability of the B cell to present the diversity of peptides from an antigen or antigenic complex required for a diverse CD4 T-cell response based on our recent studies on the role of B cells in antigen presentation to CD4 T cells in vivo (Refs 1,2 and M.J. Mamula, S. Fatehnejad and J. Craft, submitted). The activation of CD4 T cells by an exogenous protein antigen requires preseotation by a cell that can internalize the antigen, process it, and present it on the surface bound to major histocompatibility complex (MHC) class II molecules. The same APC must provide suitable costimulatory signals, the best characterized of which is the CD28 ligand B7/BB1 ~'a. Much recent data supports a role for dendritic cells (DC) in the initiation of CD4 T-cell responses to protein antigens in vWO s-7. However, earlier studies suggested that B cells were important for the induction of recall proliferative responses to protein antigens, in that mice treated from ~-;'h --';'~ anti-la ~'-: . . . . :,._a.. o-co. . . . . . . . . . . .s~a~t~ a ~ t ~ u o u y tO suppress n __,, development had defective T-cell proliferative responses that could be restored with antigen-specific B cells 8-1°. It is important to note, however, that several studies have shown that such B-cell deficient mice have normal levels of MHC class II restricted CD4 helper T cells n. Thus, these studies suggest that B cells are required for some but not all aspects of CD4 T-cell priming. Dendritic cells have been shown to be the most potent stimulators of CD4 T-cell responses to non-self MHC molecules 5, and as they constitutively express B7/BB112 and high levels of MHC class II molecules s, they are also excellent candidates for initiating CD4 T-cell responses to extrinsic antigens. However, DC are quite inefficient at antigen uptake and lack a means for discriminating between extrinsic protein antigens and self proteins whose peptides compete with them for presentation. As it is estimated that 100-200 MHC class II peptide complexes are required to signal a CD4 T cell, and as a DC has around 10 s MHC class II molecules, a maximum of 1000 peptides could be presented by such a cell. Moreover, direct evidence shows that some peptides are very much overexpressed on DC ~3, suggesting even less complexity. Thus, it seems likely that extrinsic antigens will be presented relatively inefficiently by DC, consistent with the very high protein doses required for effective presentation of peptides by the MHC class II molecules of DC 6. Thus, only those peptides that bind extremely well to the MHC class II alleles of the DC are likely to achieve levels on the cell surface sufficient to prime a helper CD4 T cell. This probably accounts for the phenomenon of immunodominant peptide epitopes in primary CD4 T-cell responses. Once helper CD4 T cells are primed to immunodominant peptides on DC, they will in turn activate specific antigen-binding B cells that internalize antigen 10 000-fold more efficiently than non-specific cell¢ 41~. When these antigen-binding B cells are activated by the primed antigen-specific helper T cells, they become competent to prime naive CD4 T cells. Because of the large amounts of protein internalized by the antigen-specific B cell, a much greater range of peptides from the original protein, or from proteins associated with it, can be presented to these naive CD4 T cells. Thus, the antigen that binds to the B cell can diversify the CD4 T-cell response. Recent studies from Eynon and Parker I~ and from Fuchs and Matzinger ~7 demonstrate that when resting B cells present antigen to CD4 T cells, tolerance resu!ts, an observation thought to reflect the absence of co-stimuiatory activity on resting B cells. By contrast, B cells activated with a variety of microbial substances can stimulate naive CD4 T-cell proliferative responses in vitro ~s, although Fuchs and Matzinger ~ find that even LPS-acti-