Long-term Memory CD8 Research Articles

Chemokine Receptor Requirements for Epidermal T-Cell Trafficking

Inflamed skin contains CD4 T-cell subsets that express chemokine receptors CCR4, CCR6, and/or CCR10. Prior attempts to reveal the distinct role(s) of each receptor in T-cell trafficking to skin have not produced a coherent story. Different conclusions drawn by separate research groups are difficult to reconcile because of the disparate inflammation models used. Here we directly compare CD4 T cells from wild-type, CCR4(-/-), CCR6(-/-), and CCR10(-/-) mice in parallel assays of trafficking to skin. Our models require direct competition between wild-type and receptor-deficient populations for access to inflamed cutaneous sites. Major histocompatibility complex-peptide tetramers allowed us to identify antigen-specific endogenous long-term memory CD4 T cells within skin after multiple topical immunizations. We separately analyzed cells from the dermal and epidermal layers, allowing us to assess the involvement of each receptor in trafficking between dermis and epidermis. We found that CCR4 deficiency reduces accumulation of memory CD4 T cells in skin by approximately 20-fold, but neither CCR6 nor CCR10 deficiency yielded any detectable effects. Strikingly, no differences in dermal versus epidermal localization were observed for cells lacking any of these three receptors. Our findings raise the possibility that CCR6 and CCR10 play (as yet) unknown roles in cutaneous T-cell immunology, unrelated to skin-specific trafficking.

CD4+ TH1 Cells Generated by Ii-PADRE DNA at Prime Phase Are Important to Induce Effectors and Memory CD8+ T Cells

The requirement for CD4 T cells in priming and maintaining cytotoxic T-lymphocyte responses presents a long-standing paradox in cellular immunology. In this study, we used sequential coadministration of a DNA vaccine encoding an invariant (Ii) chain in which the class II-associated Ii-peptide region is replaced with CD4 T-helper epitope, PADRE [Pan human leukocyte antigen-DR reactive epitope (Ii-PADRE)] or Bcl-xL with a DNA vaccine encoding Sig/E7/LAMP-1 to verify the role of CD4 T cells for the generation of effectors and memory E7-specific CD8 T-cell immune responses. Sequential vaccination, with Ii-PADRE+Sig/E7/LAMP-1 priming followed by Bcl-xL+Sig/E7/LAMP-1 boosting led to generation of E7-specific CD8 T cells, and was nearly equivalent in effect to coadministration with Ii-PADRE+Sig/E7/LAMP-1 or Bcl-xL+Sig/E7/LAMP-1 at both prime and boost. The mice vaccinated with the Ii-PADRE+Sig/E7/LAMP-1 prime-Bcl-xL+Sig/E7/LAMP-1 boost regimen exhibited better long-term E7-specific immune responses and tumor prevention effects in vivo than the mice vaccinated with the reverse sequential coadministration. After CD4 T-cell depletion, mice primed with Ii-PADRE+Sig/E7/LAMP-1 generated low numbers of E7-specific CD8 T cells and suppressed long-term memory CD8 T-cell response regardless of the sequence or combination of DNA vaccines administered. Mice primed with Bcl-xL+Sig/E7/LAMP-1 only suppressed long-term memory CD8 T-cell response after depletion of CD4 T cells before priming. Our findings suggest that activated CD4 T cells at prime phase are important to generate the antigen-specific CD8 T-cell immune responses and CD4 T cells, which are naive or activated, play a role to maintain the long-term memory responses.

A programmed gene expression initiated by clearance of infection turns CD4 memory T cells dormant (85.18)

Abstract Memory T cells survive throughout the lifetime of an individual and are protective upon recall. We have demonstrated that low levels of antigen present at the resolution of infection and upon presentation by B cells induce a state of unresponsiveness in specific CD4+ memory T cells. We have shown that this process occurs naturally during the fall of antigen. The quiescent memory T cells become activated by stimulatory signals generated by a subsequent infection. Here we show that the quiescent memory T cells survive for over a year in the absence of homeostatic proliferation. Microarray gene expression assay of long-term memory CD4 T cells revealed downregulation of proliferation, immune activation and apoptosis prevention genes in unresponsive memory CD4 T cells, compared to the responsive group. Upregulated genes in quiescent memory T cells appear to work synergistically with the downregulated genes in that group. Data from these experiments suggest that memory CD4 T cells survive in the absence of antigen for extended period of time by becoming quiescent naturally as the infection fades and resume their activity once they encounter antigen simultaneously with danger signal, providing quick and efficient immune response.

Viral antigen and extensive division maintain virus-specific CD8 T cells during chronic infection

Efficient maintenance of memory CD8 T cells is central to long-term protective immunity. IL-7– and IL-15–driven homeostatic proliferation is essential for long-term memory CD8 T cell persistence after acute infections. During chronic infections, however, virus-specific CD8 T cells respond poorly to these cytokines. Yet, virus-specific CD8 T cells often persist for long periods of time during chronic infections. We have addressed this apparent paradox by examining the mechanism for maintaining virus-specific CD8 T cells during chronic infection. We find that homeostatic cytokines (e.g., IL-7/15), inflammatory signals, and priming of recent thymic emigrants are not sufficient to maintain virus-specific CD8 T cells over time during chronic infection. Rather, our results demonstrate that viral peptide is required for virus-specific CD8 T cell persistence during chronic infection. Moreover, this viral antigen-dependent maintenance results in a dramatically different type of T cell division than is normally observed during memory T cell homeostasis. Rather than undergoing slow, steady homeostatic turnover during chronic viral infection, CD8 T cells undergo extensive peptide-dependent division, yet cell numbers remain relatively stable. These results indicate that antigen-specific CD8 T cell responses during persisting infection are maintained by a mechanism distinct from that after acute infection.

Bcl6 is essential for the generation of long-term memory CD4+ T cells

Bcl6 plays a role in the generation and maintenance of memory CD8(+) T cells. We analyzed here a role for Bcl6 in the generation of long-term memory CD4(+) T cells. Naive CD45RB(+) CD4(+) T cells from Bcl6-deficient DO11.10 (KJ1.26(+)) transgenic mice were transferred into BALB/c mice and immunized with ovalbumin peptide and LPS. Long-term memory KJ1.26(+) CD4(+) T cells from wild-type mice were detected in the spleen, lungs and liver during 10 weeks after immunization; however, Bcl6-deficient KJ1.26(+) CD4(+) T cells were vanished completely in those organs 4 weeks after immunization. Since memory CD4(+) T cells can be generated from effector CD4(+) T cells, properties of Bcl6-deficient effector CD4(+) T cells were compared with those wild-type effector CD4(+) T cells 10 days after immunization. Numbers of IFN-gamma-non-producing CD45RB(-), CD62L(+) or IL-7Ralpha(+) effector CD4(+) T cells in the spleen, lungs and liver were similar between Bcl6-deficient and wild-type CD4(+) T cells. However, the percentage of apoptotic cells in Bcl6-deficient effector CD4(+) T cells was higher than that in wild-type effector CD4(+) T cells. At the late effector phase, the number of IFN-gamma-non-producing cells and the percentage of apoptotic cells in Bcl6-deficient CD4(+) T cells were smaller and higher than those in wild-type CD4(+) T cells, respectively. These data suggest that Bcl6 in CD4(+) T cells plays a role in protection of memory precursor CD4(+) T cells from apoptosis and may involve in survivability of long-term memory CD4(+) T cells.

The adjuvant effects of co‐stimulatory molecules on cellular and memory responses to HBsAg DNA vaccination

Because DNA vaccines on their own tend to induce weak immune responses in humans, adjuvant methods are needed in order to improve their efficacy. The co-stimulatory molecules 4-1BBL, OX40L, and CD70 have been shown to induce strong T cell activities; therefore, in this study, we investigated whether they may be used as molecular adjuvants for a hepatitis B surface antigen (HBsAg) DNA vaccine (pcDS2) in eliciting strong cellular and memory responses. Compared to mice immunized with pcDS2 alone, addition of the co-stimulatory molecules increased T cell proliferation and an HBsAg-specific antibody response that was marked with a higher ratio of IgG2a/IgG1. Importantly, pcDS2 plus these co-stimulatory molecules elicited a higher level of IFN-gamma and IL-4 in CD4(+) T cells and a higher level of IFN-gamma in CD8(+) T cells. In addition, a significantly robust antigen-specific cytotoxic T lymphocyte (CTL) response and the production of long-term memory CD8(+) T cells were also observed in the groups immunized with pcDS2 plus 4-1BBL, OX40L, or CD70. Consistently, as late as 100 days after immunization, upregulated expressions of BCL-2, Spi2A, IL-7Ra, and IL-15Ra were still observed in mice immunized with pcDS2 plus these co-stimulatory molecules, suggesting the generation of memory T cells in these groups. Together, these results suggest that the co-stimulatory molecules 4-1BBL, OX40L, or CD70 can enhance the immunogenicity of HBsAg DNA vaccines, resulting in strong humoral, cellular, and memory responses. This approach may lead to an effective therapeutic vaccine for chronic hepatitis B virus (HBV) infection.

Persistence of High Viral Load Abrogates Long-Term HIV-Specific Memory CD4 T-Cells Producing Interleukin-2.

Virus-specific CD4 T-cells are believed to play a critical role in determining the persistence of memory and effector CD8 T-cell responses during viral infections. However, the reasons for which human immunodeficiency virus (HIV)-specific CD4 cells fail to generate effective CD8 cell responses remain incompletely understood. In this study, we analyzed the HIV-specific CD4 cells in 10 aviremic (viral load < 50 copies/ml) and 8 viremic (mean viral load 45,295 copies/ml) patients treated during primary HIV infection and followed for up to 6 years. At the time of apheresis, median CD4 T cell counts for aviremic and viremic patients were 671 cells/μl and 485 cells/μl respectively. Using Gag and Nef overlapping HIV peptides, the highly sensitive CFSE-based proliferation assay and intracellular staining techniques, we observed that proliferative Gag and Nef peptide-specific CD4 cell responses were 30-fold higher in aviremic patients compared to viremic. Several subsets of HIV-specific memory CD4 cells endowed with different proliferative and functional capacities were identified. We observed two subsets of HIV-specific memory CD4 cells in aviremic patients, CD45RA− CCR7+ central memory cells (Tcm) producing exclusively IL-2 and CD45RA− CCR7− effector memory cells (Tem) that produced both IL-2 and IFN-γ. In contrast, in viremic patients, Tem were found to unexpectedly produce IFN-γ exclusively. Longitudinal data showed that only cells, which were capable of producing IL-2, persisted as long-term memory cells. In viremic patients, HIV-specific CD4 cells that produce INF-γ were found only during periods of elevated viral loads. To test if the presence of IL-2 could restore the proliferation of these cells, we stimulated CD4 cells from viremic patients with a pool of peptides, which gave strong IFN-γ responses in these patients in the presence of exogenous IL-2. The addition of IL-2 during the in vitro peptide stimulation dramatically increased the fraction of proliferating cells. This experiment strongly suggests that the impaired proliferation of CD4 cells from viremic patients is not caused by a virus-mediated destruction of proliferating cells but a lack of producing IL-2. Altogether, these results indicate that long-term memory HIV-specific CD4 cell produce mainly IL-2, while those producing IFN-γ are short-lived. These findings favor a model in which Tcm are continuously produced in limited numbers but under continuous viral stimulation are rapidly induced to differentiate into IFN-γ only-producing cells that lack the capacity for self-renewal.These findings also indicate that treatment strategies aimed to increase the long-term memory CD4 cells are needed for future HIV vaccine development.

Gp100(209-2M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells.

Thirty-five HLA-A2(+) patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100(209-2M), emulsified in Montanide adjuvant. Direct ex vivo gp100(209-2M) tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer(+) CD8(+) T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100(209-2M) in vitro activation showed that for all of the patients studied tetramer(+) CD8(+) T cells produced IFN-gamma; however, some patients had significant numbers of tetramer(+) IFN-gamma(-) CD8(+)T cells suggesting functional anergy. Additionally, 8 day gp100(209-2M) in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer(+) CD8(+) T cells from postvaccine cells for 34 patients, and these IVS tetramer(+) CD8(+) T cells were functionally responsive by IFN-gamma cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8(+) T cells demonstrated the proliferation of functionally anergic gp100(209-2M)- tetramer(+) CD8(+) T cells in several patients and also indicated interpatient variability of gp100(209-2M) stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer(+) CD8+ T cells (78.1 +/- 4.2%) had either an "effector" (CD45 RA(+)/CCR7(-)) or an "effector-memory" phenotype (CD45RA(-)/CCR7(-)). Notably, analysis of PBMCs collected 12-24 months after vaccine therapy demonstrated the durable presence of gp100(209-2M)-specific memory CD8(+) T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8(+) T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.

Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against leishmania major infection.

CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major–specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.

Induction of long-term memory CD8(+) T cells for recall of viral clearing responses against influenza virus.

Induction of cytotoxic T-cell-mediated virus-clearing responses by influenza virus T cell determinant-containing peptide immunogens was examined. The most potent synthetic immunogens for eliciting pulmonary viral-clearing responses contained peptides representing determinants for CD4 and CD8 T cells (TH and CTL peptides, respectively) together with two or four palmitic acid (Pal) groups. Inoculated in adjuvant, these Pal2- or Pal4-CTL-TH lipopeptides and the nonlipidated CTL peptide induced equivalent levels of cytolytic activity in the primary effector phase of the response. The ability to recall lytic responses, however, diminished much more rapidly in CTL peptide-primed than in lipopeptide-primed mice. By 15 months postpriming, the recalled lytic activity in lipopeptide-inoculated mice remained potent, but the response induced by the CTL peptide was weak. Enumeration of specific gamma interferon-secreting CD8 T cells revealed that a greater number of these T cells had entered or remained in the memory pool in lipopeptide-primed mice, arguing for a quantitative rather than qualitative enhancement of the response on recall. Addition of either the lipid or the TH peptide to the CTL peptide was not sufficient to provide these long-lived antiviral responses, but inclusion of both components augmented the response. CD4 T cells elicited by the lipopeptides did not influence the rate of viral clearance upon challenge and most likely had a role in induction or maintenance of the memory response. It therefore appears that the lipopeptide immunogens, although not significantly superior at inducing primary effector CD8 T cells, elicit a much more effective memory population, the recall of which may account for their superiority in inducing pulmonary protection after viral challenge.

CD4+CD28- costimulation-independent T cells in multiple sclerosis.

Multiple lines of evidence suggest that CD4+ lymphocytes initiate autoimmune responses against myelin antigens in multiple sclerosis (MS). The increased frequency of activated myelin-specific cells in MS patients indicates that the activation of autoreactive cells represents a central event in the pathogenesis of the disease. We identified a CD4+ subpopulation that is characterized phenotypically by the persistent absence of surface CD28 expression and functionally by CD28-independent activation and Th1 cytokine secretion. Owing to their costimulation-independent activation and their expression of a full agonist signaling activation pattern, CD4+CD28- cells have the potential to initiate autoimmune responses in the central nervous system, a compartment devoid of professional antigen presenting cells. Long-term memory CD4+CD28- cells produce high amounts of IFN-gamma and maximally upregulate IFN-gamma and IL-12Rbeta2 chain expression in the absence of costimulation. They exhibit prominent growth characteristics and increased survival after activation, likely related to their persistent lack of CTLA-4 surface expression. The CD4+CD28- population is expanded in a subgroup of MS patients. Myelin basic protein-specific cells detected in this cell subset may play an important role in the inflammatory response within the central nervous system.

Potent induction of long-term CD8+ T cell memory by short-term IL-4 exposure during T cell receptor stimulation

An important goal of vaccination is to achieve long-term survival of functional memory T cells. Using a MHC-compatible adoptive transfer system, we show here that a short, 3-day IL-4 but not IL-2 or IL-12 exposure during in vitro T cell receptor stimulation of naive CD8(+) T cells induced long-lasting in vivo memory. Such long-term memory CD8(+) T cells expressed antigen-specific cytotoxicity and the potential for IFN-gamma and IL-4 production. Our results support the concept that functional T cell longevity can be regulated by cytokines during initial antigen encounter and provide a rational foundation for vaccine development. They also may have implications in formulating optimal therapeutic regimens of ex vivo expanded autologous cancer- and HIV-specific CD8(+) T cells. In addition, the availability of large numbers of memory CD8(+) T cells generated through our high-efficiency system should facilitate progress in the molecular dissection of CD8(+) T cell memory development.

Potent induction of long-term CD8+ T cell memory by short-term IL-4 exposure during T cell receptor stimulation.

An important goal of vaccination is to achieve long-term survival of functional memory T cells. Using a MHC-compatible adoptive transfer system, we show here that a short, 3-day IL-4 but not IL-2 or IL-12 exposure during in vitro T cell receptor stimulation of naive CD8(+) T cells induced long-lasting in vivo memory. Such long-term memory CD8(+) T cells expressed antigen-specific cytotoxicity and the potential for IFN-gamma and IL-4 production. Our results support the concept that functional T cell longevity can be regulated by cytokines during initial antigen encounter and provide a rational foundation for vaccine development. They also may have implications in formulating optimal therapeutic regimens of ex vivo expanded autologous cancer- and HIV-specific CD8(+) T cells. In addition, the availability of large numbers of memory CD8(+) T cells generated through our high-efficiency system should facilitate progress in the molecular dissection of CD8(+) T cell memory development.

CD4+ T cell priming accelerates the clearance of Sendai virus in mice, but has a negative effect on CD8+ T cell memory.

Current vaccines designed to promote humoral immunity to respiratory virus infections also induce potent CD4+ T cell memory. However, little is known about the impact of primed CD4+ T cells on the immune response to heterologous viruses that are serologically distinct, but that share CD4+ T cell epitopes. In addition, the protective capacity of primed CD4+ T cells has not been fully evaluated. In the present study, we addressed these two issues using a murine Sendai virus model. Mice were primed with an HN421-436 peptide that represents the dominant CD4+ T cell epitope on the hemagglutinin-neuraminidase (HN) of Sendai virus. This vaccination strategy induced strong CD4+ T cell memory to the peptide, but did not induce Abs specific for the Sendai virus virion. Subsequent Sendai virus infection of primed mice resulted in 1) a substantially accelerated virus-specific CD4+ T cell response in the pneumonic lung; 2) enhanced primary antiviral Ab-forming cell response in the mediastinal lymph nodes; and 3) accelerated viral clearance. Interestingly, the virus-specific CD8+ T cell response in the lung and the development of long-term memory CD8+ T cells in the spleen were significantly reduced. Taken together, our data demonstrate that primed CD4+ T cells, in the absence of pre-existing Ab, can have a significant effect on the subsequent immune responses to a respiratory virus infection.

Mucociliary Clearance in the Trachea

Tracheal mucociliary clearance is governed by the interplay between ciliary activity and secretory functions of the epithelium. These functions are altered transiently by various physiologic and injurious stimuli, and chronically in asthma, chronic bronchitis, cystic fibrosis, and other forms of airway disease. In general, these conditions are associated with a depression in mucociliary clearance that may predispose to respiratory infections and the accumulation of secretions.