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Homeostatic proliferation 27 and heterologous immunity cross-reactivity between Ags and environmental pathogens of memory T lymphocytes 28 have been identified as a major barrier of tolerance induction by costimulatory blockade, in particular in monkey models 7 , 8 , Memory T lymphocyte reactivation was therefore considered as less dependent or indeed independent of costimulation, in particular for the CD28 pathway. CTLA-4— and PD-L1—dependent mechanisms were shown to play a key role in the stability of the immune synapse and on the velocity and motility of memory T lymphocytes.

We first purified human naive and memory T lymphocytes from healthy volunteers and evaluated their proliferation after stimulations with alloantigens or with 55 HLA class I— and class II—restricted peptides from CMV, EBV, influenza virus, and tetanus toxin. We found that selective blockade of CD28 in vitro is as effective on memory as it is on naive T cells to prevent proliferation induced by either alloantigens or viral peptides.

This is in accordance with a recent study reporting that CTLA4-Ig efficiency decreased in increasingly matured human T cells when stimulated with a CMV peptide or alloantigens We investigated the CD28 requirement of Ag-specific reactivation of memory immune responses using a previously described DTH model in baboon This is suggestive of an induced immune regulation. However, we did not identify the exact mechanism: In this study, we did not observe important T cell infiltrates in skin biopsies after selective CD28 blockade, suggesting that if regulatory T cells directly control effector T cell activation, they would preferably be located in draining lymph nodes.

Finally, CD28 was also described to regulate memory T lymphocytes trafficking to extralymphoid tissue 33 , and recently it was reported that selective CD28 blockade with FR prevents skin allograft rejection in humanized mice by suppressing cutaneous lymphocyte—associated Ag CLA expression presentation by Issa et al.

If so, it is possible that FR modified immunologically challenged memory T cells in such a way that they became unable to migrate into the skin. These observations are reinforced by previous studies showing that another anti-CD28 mAb FK reduced T cell—mediated skin allograft rejection in humanized mice and reduced epidermis thinning as well as lymphocyte infiltrates of human psoriasis plaques transplanted to SCID mice 34 , We also explored the requirement of CD28 costimulation for memory humoral T-dependent immune responses using the selective monovalent CD28 antagonist. Indeed, recent studies reported that CTLA-4 controls B cell responses by intrinsic and extrinsic mechanisms 36 — 38 , in particular at the T follicular helper cell level, a specialized subset of memory lymphocytes 39 — Although SRBC challenge is considered as a T-dependent primary humoral response model in rodents, baboons but also other Old World primates, apes, and humans are naturally immunized against anti-Gal carbohydrate residues 26 expressed by microorganisms and also other mammals e.

Indeed, as described previously 42 — 44 , baboon sera were positive for preformed xenoreactive anti-SRBC IgG before any sensitization. Similar to the control of memory cellular immune response, we found that selective blockade of CD28 dose-dependently prevented anti-SRBC IgG induction after i. However, FR did not induce immune regulation in this memory humoral response model because, after complete elimination of the drug, all animals responded normally to a second challenge.

This suggests that Ag type, its presentation, or its route of administration determines whether immune regulation will take place after Ag reactivation of memory cells. A potential adverse effect of immunosuppressed memory T cells is the reactivation of latent viral infections. In this study, we did not observe exacerbation of latent viruses over the time course of the experiment until 20 wk after treatment , even at higher treatment doses. It is possible that preformed Abs against these viruses have contributed to maintaining low viral titers even though some memory cellular immunity was probably impaired.

Indeed, our KLH experiment showed that even if selective CD28 blockade could prevent memory humoral recall response during exposure to the drug, it did not modify preformed Ig titers 46 , Furthermore, it is possible also that in primates some memory T cells in charge of viral surveillance are CDindependent as it was originally described in CD28 knockout mice, which develop normal immune response against lymphocytic choriomeningitis virus infection Nonhuman primate immunology is close but could be different from human immunology.

Our study shows that memory cellular and humoral immune responses are CDdependent in vivo in nonhuman primate models and in vitro in humans. Selective blockade of CD28 prevents in vivo reactivation of memory T lymphocytes and efficiently controls inflammatory skin responses in a preclinical model. The other authors have no financial conflicts of interest.


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Centre Hospitalier Universitaire, Nantes, France; and. Abstract Novel therapies that specifically target activation and expansion of pathogenic immune cell subsets responsible for autoimmune attacks are needed to confer long-term remission. Introduction Current systemic treatments for autoimmune diseases include those inhibiting the whole immune system with immunosuppressive drugs and those that dampen inflammation as a whole. Materials and Methods Selective CD28 antagonist: SRBC and keyhole limpet hemocyanin immunization Two months before drug administration, baboons were immunized with i.

Immunophenotyping Leukocytes were from baboon blood and prepared by RBC lysis. Results CD28 is required for human memory T cell reactivation We have recently reported that selective blockade of CD28 with FR is effective in naive nonhuman primates to prevent allograft rejection 23 and protects from acute fatal experimental autoimmune encephalomyelitis 24 , the elected experimental model of multiple sclerosis. Selective CD28 blockade tackles Ag-specific memory skin inflammatory response To investigate the role of CD28 in vivo on memory T cell reactivation, we first used a DTH model in baboons, having demonstrated that expression of CD28 on baboon memory T cells has a similar pattern to humans, which is not the case for macaques 9 , Selective CD28 blockade controls humoral memory recall response without disruption of preformed immunity To investigate the role of CD28 in vivo on memory T-dependent humoral responses, we first used the T-dependent SRBC immunization model.

Discussion Our study questioned the requirement of CD28 costimulation for memory T lymphocyte reactivation at the preclinical level.

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Cytokine control of memory T-cell development and survival. Memory T cells need CD28 costimulation to remember. Duration of TCR stimulation determines costimulatory requirement of T cells. T cell activation determined by T cell receptor number and tunable thresholds.

Induction and visualization of mucosal memory CD8 T cells following systemic virus infection. Alefacept promotes co-stimulation blockade based allograft survival in nonhuman primates. Kawai , et al. Host alloreactive memory T cells influence tolerance to kidney allografts in nonhuman primates. Le Bas-Bernardet , V.

Charpy , et al. Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD A more selective costimulatory blockade of the CDB7 pathway. Alternatives to calcineurin inhibition in renal transplantation: In some of the experiments with unfixed cells, dead cells were excluded by staining with propidium iodide. All data were analyzed with FlowJo software Tree Star. Doublet discrimination was applied for cell cycle analysis. Percoll gradient separation was performed as described 30 , Briefly, a discontinuous After centrifugation, cells were retrieved from three interfaces: We assessed the minimum time needed in our hands to cause resting murine T cells to proliferate when stimulated with plate-bound anti-CD3 and anti-CD28 Abs.

Stimulation was stopped by transferring cells to uncoated plates 12 , 27 , 32 , and proliferation was assessed 2—4 days later by CFSE dilution In general, 10—14 h of continuous stimulation resulted in most of the cells being equally distributed into four to five CFSE peaks by 3 days below and data not shown.

Having established minimum continuous stimulation periods required for proliferation, discontinuous periods of stimulation were used to test whether T cells can be integrate interrupted signals over time. To allow the comparison of groups whose final stimulation ended at different times, proliferation was assessed at several analyses that were each offset by 7 h. Strikingly, cells that were rested for 7 h between the two 7-h periods of stimulation proliferated to a similar extent as those whose stimulation was continuous.

When the time after the final stimulation is taken into account, the percentage of cells from the initial population that underwent one or more divisions was comparable between cells receiving continuous and those receiving discontinuous stimulation Fig. Nevertheless discontinuously stimulated cells proliferated as well as continuously stimulated cells data not shown. Therefore, naive T cells can recall a subthreshold period of stimulation hours after its termination.

The diagonal lines indicate cells that were analyzed at the same time after the last stimulation ended.

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D , The percent of cells from the original population that had divided was calculated with FlowJo software. Representative data from one of three experiments is shown. To ensure that a low level of T cell signaling did not persist during the interval of rest, proximal TCR-mediated signaling was interrupted during this period with the Src family kinase inhibitor PP2 34 , PP2 was added to the cells at the time of their transfer from Ab-coated to uncoated wells, and was removed by washing when the cells were returned to Ab-coated plates.

As expected, T cells stimulated for as long as 26 h in the presence of PP2 did not proliferate when analyzed 41 h after removing PP2 Fig. Cells that were stimulated for 5 h, rested for 20 h, and then restimulated for 5 h, in contrast, proliferated extensively by 67 h. The inclusion of PP2 during the h rest period had no effect on the extent to which the restimulated cells proliferated.

Inhibition of Src-kinase signaling during the period of rest does not prevent recollection of previous stimulation.

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One representative experiment of two is shown. To determine whether integrated responses to discontinuous stimulation occur with a physiologic ligand, APC were used to present cognate MHC-peptide to T cells of known Ag-specificity. T cells were obtained from 5C. The fibroblast cell line P The APC were allowed to phagocytose magnetic beads before their use so that they could be efficiently removed from the T cells by magnetic separation. C7 T cells for up to 18 h, resulting in only a small number of cells that had divided by 67—73 h, thereby providing background values Fig.

Stimulation with Ag-pulsed P However, when assessed 6 h later so that the time after the second stimulation was 55 h, the same as the S12 group when analyzed at 67 h, the discontinuously stimulated population had caught up and reached the same division profile as the cells that received 12 h of uninterrupted stimulation Fig. Therefore, T cells remember discrete periods of antigenic stimulation separated by periods of rest. Integration of independent stimuli occurs for antigenic stimulation. Peripheral lymph node T cells from 5C.

Stimulation was terminated by magnetic separation of T cells from APC. The diagonal line indicates cells that were stimulated for 55 h after the final stimulation ended. Duplicate samples are shown from one of three experiments. Cell cycle progression involves blast transformation, characterized by an increase in new RNA and protein synthesis and cell size, and a corresponding decrease in cell density.

When unstimulated cells were separated after 23 h of culture virtually no viable cells were recovered from the low density layer, the large majority being of high density Fig. There were some apoptotic cells low forward scatter, high side scatter present in all fractions after the overnight culture.

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The densities of the different fractions of cells after 5 h of stimulation and 18 h of rest were reflected in their forward vs side scatter profiles. Some low density cells blasts were recovered, as well as a large number of cells with high and intermediate density Fig. At 23 h, cells were separated Percoll density gradient. Forward and side-scatter of unseparated and Percoll density separated cells are shown. The Table indicates the percentage of cells recovered in the different Percoll fractions.

B , Following fractionation, cells were left untreated or subjected to a second 5-h period of stimulation, and proliferation was assessed 40 h after fractionation. CFSE dilution is shown 45 h after fractionation. One representative experiment of three is shown.


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  • To determine the proliferative capacity of the different density populations, one-half of each fraction was left untreated and the other was stimulated for 5 h. All populations were analyzed 40 h after Percoll fractionation or 63 h from the beginning of the experiment. The high density cells did not commit to proliferate whether or not they received a second period of activation, and thus reflect the population that did not remember the initial subthreshold stimulation. The most interesting group was that of the intermediate density cells.

    If they received only one 5-h period of stimulation the cells did not progress into division. However, a second 5-h stimulation caused the majority of them to proceed through the cell cycle by 40 h. Therefore, the intermediate density population comprises cells that have not been adequately stimulated to divide after one stimulus, but can sum two discontinuous periods of stimulation and respond with vigorous proliferation.

    As above, cells that had been rested for 23 h before Percoll density separation did not proceed to proliferate, whether or not they received a 6-h period of stimulation before analysis Fig. Importantly, the intermediate density cells did not proliferate after only one round of stimulation, but after 18 h of rest they responded very well to a second 6-h stimulation. Only a few high density T cells proliferated after a second stimulation, which may represent a small number of contaminating intermediate density cells.

    In any case, the results clearly show that naive T cells of intermediate density can integrate two subthreshold periods of stimulation separated by at least 18 h. The density of T cells after activation decreases as they enter the cell cycle, with cells in G 0 being the densest. RNA levels increase in a continuum from the G 0 phase to dividing cells. Immediately after fractionation, the low density population was composed almost entirely of cells that had progressed past G 0 Fig. What distinguished the intermediate from the high density cells was their RNA content, which was elevated in the former.

    Therefore, we conclude that the intermediate density cells are largely in G 1 , whereas the high density cells are in G 0. After 15 or 25 h of continuous secondary stimulation, the large majority of cells in the low density fraction were actively cycling. In contrast, after 15 h of continuous restimulation high density cells had just begun to enter G 1. Only after 25 h of restimulation did a substantial number of high density cells progress into G 1 Fig.

    Intermediate density cells are in the G 1 , and progress into the cell cycle upon restimulation faster than high density G 0 cells.

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    A , Analysis of unstimulated cells. One set of cells was analyzed immediately after fractionation first row. A second and third set was restimulated for 15 or 25 h and then immediately analyzed second and third rows. The percentage of cells in each gate is shown. C , The same cells as in B were analyzed 25 h after fractionation without 0 or with S5 a 5-h period of restimulation. Cell cycle analysis was performed on the different density fractions that had received one or two subthreshold 5 h periods of stimulation Fig. Cells in all three density populations retained their cell cycle phenotypes when simply cultured for 25 h after Percoll separation, although the low density cells showed some evidence of becoming less active compare with Fig.

    The same treatment caused a small percentage of high density G 0 cells to progress into early G 1 , but the majority remained in G 0. Therefore, both intermediate and high density fractions were able to respond to a secondary stimulus, but only the intermediate density fraction was able to respond to 5 h of restimulation by passing through the whole cell cycle. Having observed that T cells were able to recall previous stimulation at least for 20 h, experiments were performed to test the maximum duration of reset permissible Fig.

    After a 5-h subthreshold stimulation, T cells were rested for up to 46 h and restimulated another 5 h. Cells that received only one period of stimulation divided very little by 74—92 h. Samples that were rested for 20—25 h before restimulation, had divided extensively by 74 h, with a similar division profile observed at 92 h, indicating that by 72 h they had divided to their full potential.

    Therefore, cells can remain in a responsive state for at least 2 days after the initial subthreshold stimulation is withdrawn. Cells recollect stimulation for long periods of time. One set of cells was left untreated. To determine whether other activation-induced T cell responses are enhanced in G 1 T cells, IL-2 production was measured. In the experimental group, cells were stimulated for 4 h S4 and then rested for 19 h, at which time they were Percoll-fractionated and rested or restimulated for 5 h in the presence of monensin.

    It is, however, conceivable that inhibition of proliferation induced by CTLA-4 and inhibition of that induced by CD4 occur independent from each other, but signaling through both is required to reach the threshold that inhibits memory T cell proliferation. Memory CD4 T cell proliferation may therefore occur at minimal CD28 ligation as long as inhibitory signals through CD4 are not delivered.

    Given that memory T cells have a lower threshold for proliferation than naive T cells 40 , there is a danger that memory CD4 T cells might become activated by the signals for their survival. It has been shown that hyporesponsive gppositive CD4 T cells can be induced to proliferate when CD28 is ligated by mAb 41 , We propose that CTLA-4 signaling is not just required for down-regulation of already activated T cells, but that it is a critical regulator of resting memory CD4 T cells.

    Crispe for critical review of the manuscript, and Patricia Ranney for looking after the mice. Bottomly, Cedar St. We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

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    Skip to main content. J Immunol December 1, , 11 ;. Abstract Regulation of peripheral T cell responses is critical for preserving self tolerance. Antibodies The following mAbs were purified from supernatants of hybridomas maintained in this laboratory using standard protein A or protein G affinity chromatography: Results Proliferation of memory CD4 T cells to soluble anti-CD3 bound to FcRs on fibroblasts is costimulation dependent Memory CD4 T cells proliferate to plate-bound anti-CD3, while naive T cells fail to do so, suggesting that memory T cells proliferate in the absence of costimulation Discussion Activation of peripheral T cells is stringently regulated to prevent proliferation to self.

    Acknowledgments We thank Dr. Peripheral T cell survival requires continual ligation of the T cell receptor to major histocompatibility complex-encoded molecules. Expression and functional significance of an additional ligand for CTLA The role of the CD28 receptor during T cell responses to antigen. Differential activation requirements for virgin and memory T cells.

    Virgin and memory T cells have different requirements for activation via the CD2 molecule. Differential T cell receptor-mediated signaling in naive and memory CD4 T cells. Naive and memory T cells show distinct pathways of lymphocyte recirculation. Tissue-specific migration pathways by phenotypically distinct subpopulations of memory T cells.

    Naive versus memory CD4 T cell response to antigen. B7-independence of the secondary type 2 response. CTLA-4, a negative regulator of T-lymphocyte activation. The emerging role of CTLA-4 as an immune attenuator. Lymphoproliferative disorders with early lethality in mice deficient in CTLA Control of memory CD4 T cell activation: CTLA-4 can function as a negative regulator of T cell activation.

    Tyrosine phosphorylation controls internalization of CTLA-4 by regulating its interaction with clathrin-associated adaptor complex AP Van der Merwe, P. Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expressing dendritic cells.

    Rapid effector function in CD8 memory T cells.