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.     

Failure of CD4 T-cells to respond to liver-derived antigen and to provide help to CD8 T-cells

CD4 T-cell help is required for the induction of efficient CD8 T-cells responses and the generation of memory cells. Lack of CD4 T-cell help may contribute to an exhausted CD8 phenotype and viral persistence. Little is known about priming of CD4 T-cells by liver-derived antigen. We used TF-OVA mice expressing ovalbumin in hepatocytes to investigate CD4 T-cell priming by liver-derived antigen and the impact of CD4 T-cell help on CD8 T-cell function. Naïve and effector CD4 T-cells specific for ovalbumin were transferred into TF-OVA mice alone or together with naïve ovalbumin-specific CD8 T-cells. T-cell activation and function were analyzed. CD4 T-cells ignored antigen presented by liver antigen-presenting cells (APCs) in vitro and in vivo but were primed in the liver-draining lymph node and the spleen. No priming occurred in the absence of bone-marrow derived APCs capable of presenting ovalbumin in vivo. CD4 T-cells primed in TF-OVA mice displayed defective Th1-effector function and caused no liver damage. CD4 T-cells were not required for the induction of hepatitis by CD8 T-cells. Th1-effector but not naïve CD4 T-cells augmented the severity of liver injury caused by CD8 T-cells. Our data demonstrate that CD4 T-cells fail to respond to liver-derived antigen presented by liver APCs and develop defective effector function after priming in lymph nodes and spleen. The lack of CD4 T-cell help may be responsible for insufficient CD8 T-cell function against hepatic antigens.     

Alterations in the phenotype of CMV-specific and total CD8+ T-cell populations in Wegener's granulomatosis

Wegener's granulomatosis (WG) is an autoimmune disease of as yet unknown etiology. To date it has remained obscure what causes WG or determines disease progression. Case reports suggest that viral infections such as cytomegalovirus (CMV) reactivation may contribute to disease flares. In this study we found a skewing of the phenotype of CMV-specific CD8+tet(ramer)+ T-cells in WG. A marked proportion of these cells displayed a late differentiated "effector memory" T-cell phenotype with decreased expression of CD28 and CD62L, and heterogeneous CD27 expression, features which were also seen in CD8+tet- T-cells in WG, but not in controls. Our results might reflect profound generalized changes in the CD8+ T-cell compartment also affecting virus-specific T-cell responses in WG.     

CD80 and CD86 Differentially Regulate Mechanical Interactions of T-Cells with Antigen-Presenting Dendritic Cells and B-Cells

Functional T-cell responses are initiated by physical interactions between T-cells and antigen-presenting cells (APCs), including dendritic cells (DCs) and B-cells. T-cells are activated more effectively by DCs than by B-cells, but little is known about the key molecular mechanisms that underpin the particular potency of DC in triggering T-cell responses. To better understand the influence of physical intercellular interactions on APC efficacy in activating T-cells, we used single cell force spectroscopy to characterize and compare the mechanical forces of interactions between DC:T-cells and B:T-cells. Following antigen stimulation, intercellular interactions of DC:T-cell conjugates were stronger than B:T-cell interactions. DCs induced higher levels of T-cell calcium mobilization and production of IL-2 and IFNγ than were elicited by B-cells, thus suggesting that tight intercellular contacts are important in providing mechanically stable environment to initiate T-cell activation. Blocking antibodies targeting surface co-stimulatory molecules CD80 or CD86 weakened intercellular interactions and dampen T-cell activation, highlighting the amplificatory roles of CD80/86 in regulating APC:T-cell interactions and T-cell functional activation. The variable strength of mechanical forces between DC:T-cells and B:T-cell interactions were not solely dependent on differential APC expression of CD80/86, since DCs were superior to B-cells in promoting strong interactions with T-cells even when CD80 and CD86 were inhibited. These data provide mechanical insights into the effects of co-stimulatory molecules in regulating APC:T-cell interactions.     

Mesenchymal stromal cells augment CD4+ and CD8+ T-cell proliferation through a CCL2 pathway

Background aimsMesenchymal stromal cells (MSC) derived from bone marrow are immunosuppressive in vitro and in vivo. Recent evidence, however, has shown that in certain settings, MSC can also be immunostimulatory. The mechanisms involved in this process are largely unknown.MethodsMouse spleen T cells were stimulated with allogeneic mixed lymphocyte reaction (MLR) or anti-CD3/CD28 beads and treated with autologous bone marrow MSC or MSC-conditioned medium. CD4+ and CD8+ T-cell proliferation was analyzed after treatment.ResultsWe show that MSC have both suppressive and stimulatory functions toward T cells after stimulation with anti-CD3/CD28 beads or in an MLR. This depended on the ratio of MSC to responder T cells, with low numbers of MSC increasing and higher numbers inhibiting T-cell proliferation. Immunostimulatory function was mediated, in part, by soluble factors. MSC immunosuppression of the MLR was indirect and related to inhibition of antigen-presenting cell maturation. Direct effects of MSC-conditioned medium during anti-CD3/CD28 stimulated proliferation were entirely stimulatory and required the presence of the T-cell receptor. MSC supernatant contained both CCL2 and CCL5 at high levels, but only CCL2 level correlated with the ability to augment proliferation. An anti-CCL2 antibody blocked this proliferative activity.ConclusionsCCL2 plays an important role in the immunostimulatory function of MSC, and we further hypothesize that the immunomodulatory role of MSC is determined by a balance between inhibitory and stimulatory factors, suggesting the need for caution when these cells are investigated in clinical protocols.     

CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells

CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.     

Functional properties and lineage relationship of CD8+ T cell subsets identified by expression of IL-7 receptor alpha and CD62L

Three major subsets of Ag-experienced CD8+ T cells have been identified according to their expression of CD62L and CD127. These markers are associated with central memory T cells (CD62L+ CD127+), effector memory T cells (CD162L- CD127+), and effector T cells (CD62L- CD127-). In this study we characterized the development of these three populations during acute and chronic viral infections and after immunization with virus-like particles and determined their lineage relation and functional and protective properties. We found that the balance between the three subsets was critically regulated by the availability of Ag and time. After initial down-regulation of CD127, the responding CD8+ T cell population down-regulated CD62L and re-expressed CD127. Dependent on Ag availability, the cells then further differentiated into CD62L- CD127- effector cells or, in the absence of Ag, re-expressed CD62L to become central memory T cells. Although all three populations efficiently produced effector cytokines such as IFN-gamma, CD62L- CD127- effector cells exhibited the highest ex vivo lytic potential. In contrast, CD62L+ CD127+ central memory T cells most efficiently produced IL-2 and proliferated extensively in vitro and in vivo upon antigenic restimulation. Strikingly, only effector and effector memory, but not central memory, T cells were able to protect against peripheral infection with vaccinia virus, whereas central memory T cells were most potent at protecting against systemic infection with lymphocytic choriomeningitis virus, indicating that the antiviral protective capacities of specific CD8+ T cell subsets are closely related to the nature of the challenging pathogen.     

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.     

Activation of CD4+ T-lymphocytes in asymptomatic HIV infected patients induce the program action of lymphocyte death by apoptosis]

Activation-induced programmed cell death, or apoptosis, is a physiological cell suicide process involved in the negative thymic selection of the T-cell repertoire. We have proposed that the inappropriate re-emergence in the mature CD4+ T-cell population of such a death program could explain both the early dysfunction and the late depletion of CD4+ T-cells from human immunodeficiency virus (HIV)-infected individuals. We present evidence showing that the selective failure of T-cells from 10 HIV-infected asymptomatic individuals (with normal CD4+ T-cell counts) to proliferate in vitro to pokeweed mitogen and to self-major histocompatibility complex class-II T-cell receptor mobilization by superantigens is due to the induction by these stimuli of CD4+ T-cell death. This death process has characteristic features of apoptosis, including DNA fragmentation into multiples of a 200 base pair unit, and the preventive effect of the protein synthesis inhibitor cycloheximide. These findings suggest that in vivo CD4+ T-cell suicide upon activation might account, independently of any HIV-mediated cytopathic effect, for the progressive depletion of CD4+ T-cells that leads to AIDS.     

Complex memory T-cell phenotypes revealed by coexpression of CD62L and CCR7

Antigen-experienced T cells have been divided into CD62L+ CCR7+ central memory (TCM) and CD62L- CCR7- effector memory (TEM) cells. Here, we examined coexpression of CD62L and CCR7 in lymphocytic choriomeningitis virus-specific memory CD8 T cells from both lymphoid and nonlymphoid tissues. Three main points emerged: firstly, memory cells frequently expressed a mixed CD62L- CCR7+ phenotype that differed from the phenotypes of classical TEM and TCM cells; secondly, TCM cells were not restricted to lymphoid organs but were also present in significant numbers in nonlymphoid tissues; and thirdly, a major shift from a TCM to TEM phenotype was found in memory cells that had been stimulated repetitively with antigen.     

Inhibition of human immunodeficiency virus type 1 gp120 presentation to CD4 T cells by antibodies specific for the CD4 binding domain of gp120

Human immunodeficiency virus (HIV)-specific CD4 T-cell responses, particularly to the envelope glycoproteins of the virus, are weak or absent in most HIV-infected patients. Although these poor responses can be attributed simply to the destruction of the specific CD4 T cells by the virus, other factors also appear to contribute to the suppression of these virus-specific responses. We previously showed that human monoclonal antibodies (MAbs) specific for the CD4 binding domain of gp120 (gp120(CD4BD)), when complexed with gp120, inhibited the proliferative responses of gp120-specific CD4 T-cells. MAbs to other gp120 epitopes did not exhibit this activity. The present study investigated the inhibitory mechanisms of the anti-gp120(CD4BD) MAbs. The anti-gp120(CD4BD) MAbs complexed with gp120 suppressed gamma interferon production as well as proliferation of gp120-specific CD4 T cells. Notably, the T-cell responses to gp120 were inhibited only when the MAbs were added to antigen-presenting cells (APCs) during antigen pulse; the addition of the MAbs after pulsing caused no inhibition. However, the anti-gp120(CD4BD) MAbs by themselves, or as MAb/gp120 complexes, did not affect the presentation of gp120-derived peptides by the APCs to T cells. These MAb/gp120 complexes also did not inhibit the ability of APCs to process and present unrelated antigens. To test whether the suppressive effect of anti-gp120(CD4BD) antibodies is caused by the antibodies' ability to block gp120-CD4 interaction, APCs were treated during antigen pulse with anti-CD4 MAbs. These treated APCs remained capable of presenting gp120 to the T cells. These results suggest that anti-gp120(CD4BD) Abs inhibit gp120 presentation by altering the uptake and/or processing of gp120 by the APCs but their inhibitory activity is not due to blocking of gp120 attachment to CD4 on the surface of APCs.     

Expression of the IL-7 Receptor Alpha-Chain Is Down Regulated on the Surface of CD4 T-Cells by the HIV-1 Tat Protein

HIV infection elicits defects in CD4 T-cell homeostasis in both a quantitative and qualitative manner. Interleukin-7 (IL-7) is essential to T-cell homeostasis and several groups have shown reduced levels of the IL-7 receptor alpha-chain (CD127) on both CD4 and CD8 T-cells in viremic HIV+ patients. We have shown previously that soluble HIV Tat protein specifically down regulates cell surface expression of CD127 on human CD8 T-cells in a paracrine fashion. The effects of Tat on CD127 expression in CD4 T-cells has yet to be described. To explore this effect, CD4 T-cells were isolated from healthy individuals and expression levels of CD127 were examined on cells incubated in media alone or treated with Tat protein. We show here that, similar to CD8 T-cells, the HIV-1 Tat protein specifically down regulates CD127 on primary human CD4 T-cells and directs the receptor to the proteasome for degradation. Down regulation of CD127 in response to Tat was seen on both memory and naive CD4 T-cell subsets and was blocked using either heparin or anti-Tat antibodies. Tat did not induce apoptosis in cultured primary CD4 T-cells over 72 hours as determined by Annexin V and PI staining. Pre-incubation of CD4 T-cells with HIV-1 Tat protein did however reduce the ability of IL-7 to up regulate Bcl-2 expression. Similar to exogenous Tat, endogenously expressed HIV Tat protein also suppressed CD127 expression on primary CD4 T-cells. In view of the important role IL-7 plays in lymphocyte proliferation, homeostasis and survival, down regulation of CD127 by Tat likely plays a central role in immune dysregulation and CD4 T-cell decline. Understanding this effect could lead to new approaches to mitigate the CD4 T-cell loss evident in HIV infection.     

Cutting edge: L-selectin (CD62L) expression distinguishes small resting memory CD4+ T cells that preferentially respond to recall antigen

Naive CD4+ T cells use L-selectin (CD62L) expression to facilitate immune surveillance. However, the reasons for its expression on a subset of memory CD4+ T cells are unknown. We show that memory CD4+ T cells expressing CD62L were smaller, proliferated well in response to tetanus toxoid, had longer telomeres, and expressed genes and proteins consistent with immune surveillance function. Conversely, memory CD4+ T cells lacking CD62L expression were larger, proliferated poorly in response to tetanus toxoid, had shorter telomeres, and expressed genes and proteins consistent with effector function. These findings suggest that CD62L expression facilitates immune surveillance by programming CD4+ T cell blood and lymph node recirculation, irrespective of naive or memory CD4+ T cell phenotype.     

Selective killing of CD8+ cells with a 'memory' phenotype (CD62Llo) by the N-acetyl-D-galactosamine-specific lectin from Viscum album L

As reported previously by our group, among the toxic proteins from Viscum album L. only the mistletoe lectins (MLs) induce the apoptotic killing pathway in human lymphocytes. Although one may expect a homogenous distribution of carbohydrate domains on cell surface receptors for the carbohydrate binding B chains of the toxic protein, the sensitivity of cells to these B chains obviously differ. Here we report a selective killing of CD8+ CD62Llo cells from healthy individuals by the galNAc-specific ML III (and RCA60, which binds to gal and galNAc), while the gal-specific ML I was less effective. This selective killing is not sufficiently explained by protein synthesis inhibition alone, since this subset was not affected by other ribosome inhibiting proteins such as the lectin from Ricinus communis (RCA120), lectin from Abrus precatorus (APA), abrin A, and inhibitors of RNA, DNA and/or protein synthesis such as actinomycin D, mitomycin C, and cycloheximide. We conclude that CD8+ cells with 'memory' phenotype (CD62Llo) are more sensitive to the ML III-mediated killing than their CD8+ CD62Lhi counterparts, CD4+ T cells, and CD19+ B cells. These cells probably express a distinct receptor with galNAc domains that is missing or not active on CD8+ cells with a 'naive' phenotype.     

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease

The adoptive transfer of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.     

CD8+ T-cell mediated anti-tumor responses cross-reacting against 9L and RT2 rat glioma cell lines

We have shown that vaccination of animals with two distinct commonly used glioma cell lines, 9L and RT2, generated cross-reactive cellular anti-tumor immunity. Peripheral vaccination with either cell line 9L or RT2 resulted in MHC Class I restricted effector cells capable of in vitro cytolytic activity against both target 9L and RT2 cells but not the syngeneic F98 glioma cell line. In vitro cross-reactive cytolytic activity could be measured for as long as 6 months from the time of initial vaccination. Fractionation of splenic effector cells revealed the cytolytic activity to be CD8+ T-cell mediated but required CD4+ T-cells for effective antigen presentation. Anti-tumor immunity generated after vaccination with either 9L or RT2 was completely protective against subsequent subcutaneous inoculation of animals with either 9L or RT2 cells and resulted in prolonged survival in animals inoculated intracranially with either cell line. Our results suggest that despite the different methods used in their derivation, 9L and RT2 glioma cells share a common glioma antigen recognized by the cellular arm of the immune response.     

Internalization of CD4 molecules in human T-cells demonstrated by immuno-electron microscopy.

Internalization of CD4 molecules on human CD4-enriched T-cells was demonstrated by immunocytochemical electron microscopy. CD4+ T-cell subclones were obtained from normal human peripheral blood, followed by one-way MLC screening and co-culturing with IL-2. Fixed and non-fixed T-cell samples were indirectly immunolabeled with mouse anti-human CD4 monoclonal antibody and goat anti-mouse IgG conjugated with peroxidase. Unfixed T-cells were immunolabeled at 4 degrees C and then re-incubated for 5-45 min at 37 degrees C. The selected CD4+ T-cell subclones showed strong CD4 binding on the cell surface after IL-2 incubation. However, fresh T-cells, monocytes, bone marrow cells and CD8+ T-cells all stained negative for CD4. The distribution of CD4 molecules on the fixed cell surface showed a homogeneous pattern. Capping and internalization of CD4-antibody-peroxidase complexes from the cell surfaces were observed follow a pathway of receptor-mediated endocytosis in unfixed T cells. Endocytotic vesicles, vacuoles of diverse sizes and shapes near the cell membrane or deep in the cell center were found to contain CD4 molecules. Negatively stained Golgi saccules were observed up to 45 min after re-incubation. These results suggest that increased CD4 molecules can be induced on the surface of normal human T-cells in vitro. Internalization and accumulation of CD4 molecules occurred in CD4-enriched T-cells with IL-2 pretreatment.     

TLR4/MYD88-dependent, LPS-induced synthesis of PGE2 by macrophages or dendritic cells prevents anti-CD3-mediated CD95L upregulation in T cells

Antigen-presenting cells (APCs) control T-cell responses by multiple mechanisms, including the expression of co-stimulatory molecules and the production of cytokines and other mediators that control T-cell proliferation, survival and differentiation. Here, we demonstrate that soluble factor(s) produced by Toll-like receptor (TLR)-activated APCs suppress activation-induced cell death (AICD). This effect was observed in non-stimulated APCs, but it was significantly increased after lipopolysaccharide (LPS) treatment. Using different KO mice, we found that the LPS-induced protective factor is dependent on TLR4/MyD88. We identified the protective factor as prostaglandin E(2) (PGE(2)) and showed that both APC-derived supernatants and PGE(2) prevented CD95L upregulation in T cells in response to TCR/CD3 stimulation, thereby avoiding both AICD and activated T cell killing of target macrophages. The PGE(2) receptors, EP2 and EP4, appear to be involved since pharmacological stimulation of these receptors mimics the protective effect on T cells and their respective antagonists interfere with the protection induced by either APCs derived or synthetic PGE(2). Finally, the engagement of EP2 and EP4 synergistically activates protein kinase A (PKA) and exchange protein directly activated by cAMP pathways to prevent AICD. Taken together, these results indicate that APCs can regulate T-cell levels of CD95L by releasing PGE(2) in response to LPS through a TLR4/MyD88-dependent pathway, with consequences for both T cell and their own survival.     

Homogenization of TCR repertoires within secondary CD62Lhigh and CD62Llow virus-specific CD8+ T cell populations

Influenza virus-specific CD8(+) T cell clonotypes generated and maintained in C57BL/6J mice after respiratory challenge were found previously to distribute unequally between the CD62L(low) "effector" (T(EM)) and CD62L(high) "central" (T(CM)) memory subsets. Defined by the CDR3beta sequence, most of the prominent TCRs were represented in both the CD62L(high) and CD62L(low) subsets, but there was also a substantial number of diverse, but generally small, CD62L(high)-only clonotypes. The question asked here is how secondary challenge influences both the diversity and the continuity of TCR representation in the T(CM) and T(EM) subsets generated following primary exposure. The experiments use single-cell RT-PCR to correlate clonotypic composition with CD62L phenotype for secondary influenza-specific CD8(+) T cell responses directed at the prominent D(b)NP(366) and D(b)PA(224) epitopes. In both the acute and long-term memory phases of the recall responses to these epitopes, we found evidence of a convergence of TCR repertoire expression for the CD62L(low) and CD62L(high) populations. In fact, unlike the primary response, there were no significant differences in clonotypic diversity between the CD62L(low) and CD62L(high) subsets. This "TCR homogenization" for the CD62L(high) and CD62L(low) CD8(+) populations recalled after secondary challenge indicates common origin, most likely from the high prevalence populations in the CD62L(high) central memory set. Our study thus provides key insights into the TCR diversity spectrum for CD62L(high) and CD62L(low) T cells generated from a normal, unmanipulated T cell repertoire following secondary challenge. A better understanding of TCR selection and maintenance has implications for improved vaccine and immunotherapy protocols.     

Leishmania donovani: role of CD2 on CD4+ T-cell function in Visceral Leishmaniasis

In this study, we investigated whether alteration in the CD2 mediated coordination of an immune response was associated with down regulation of CD4 associated Th1 cell response during Visceral Leishmaniasis (VL). Leishmania donovani (Ld) infection in VL patients markedly reduced expression of CD2 cell surface antigen on CD4+ cells. T-cells of VL patients were mostly in G0/G1 stage of the cell cycle (98.20%) with little or no activity of protein kinase C-alpha (PKC-alpha) isoform. However, pre-incubation with activating anti-CD2 monoclonal antibody (MAb) resulted in a corresponding increase up to 2.52-fold in T-cells of G2/M population supported by both activity and expression of PKC-alpha isoform. Furthermore, we observed that co-incubation of T-cell with anti-CD2 increased the lymphocyte-blast population in patients in whom the CD4 cells became more antigen responsive (CD4+ CD69+ cells). Consistent with these observations, it was shown that 59.3% of CD4 cells from patients responded to Ld by producing IFN-gamma. Even in the culture condition, when the T-cells from patients were depleted of APC, IFN-gamma production was noticed after CD2 activation. On the other hand, IL-4 production became low in the anti-CD2 antibody supplemented peripheral blood mononuclear cells (PBMNCs) culture. These findings imply that infection with L. donovani induces less CD2 on the surface of CD4+ T-cells, which once activated orchestrate the protective IFN-gamma dominant host defense mechanism via PKC-mediated signal transduction and cell cycle.