Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL

Experimental data suggest that negative selection of thymocytes can occur as a result of supraoptimal antigenic stimulation. It is unknown, however, whether, such mechanisms are at work in mature CD8+ T lymphocytes. Here, we show that CD8+ effector cytotoxic T lymphocytes (CTL) are susceptible to proliferative inhibition by high dose peptide antigen, leading to apoptotic death mediated by TNF-α release. Such inhibition is not reflected in the cytolytic potential of the CTL, since concentrations of antigen that are inhibitory for proliferation promote efficient lysis of target cells. Thus, although CTL have committed to the apoptotic pathway, the kinetics of this process are such that CTL function can occur before death of the CTL. The concentration of antigen required for inhibition is a function of the CTL avidity, in that concentrations of antigen capable of completely inhibiting high avidity CTL maximally stimulate low avidity CTL. Importantly, the inhibition can be detected in both activated and resting CTL. Blocking studies demonstrate that the CD8 molecule contributes significantly to the inhibitory signal as the addition of anti-CD8 antibody restores the proliferative response. Thus, our data support the model that mature CD8+ CTL can accommodate an activation signal of restricted intensity, which, if surpassed, results in deletion of that cell

Expression of the HPV16E7 Oncoprotein by Thymic Epithelium is Accompanied by Disrupted T Cell Maturation and a Failure of the Thymus to Involute with Age

Transgenic mice expressing the E7 protein of HPV16 from the keratin 14 promoter demonstrate increasing thymic hypertrophy with age. This hypertrophy is associated with increased absolute numbers of all thymocyte types, and with increased cortical and medullary cellularity. In the thymic medulla, increased compartmentalization of the major thymic stromal cell types and expansion of thymic epithelial cell population is observed. Neither an increased rate of immature thymocyte division nor a decreased rate of immature thymocyte death was able to account for the observed hypertrophy

Modulation of antigen presenting cell functions during chronic HPV infection.

High-risk human papillomaviruses (HR-HPV) infect basal keratinocytes, where in some individuals they evade host immune responses and persist. Persistent HR-HPV infection of the cervix causes precancerous neoplasia that can eventuate in cervical cancer. Dendritic cells (DCs) are efficient in priming/cross-priming antigen-specific T cells and generating antiviral and antitumor cytotoxic CD8+ T cells. However, HR-HPV have adopted various immunosuppressive strategies, with modulation of DC function crucial to escape from the host adaptive immune response. HPV E6 and E7 oncoproteins alter recruitment and localization of epidermal DCs, while soluble regulatory factors derived from HPV-induced hyperplastic epithelium change DC development and influence initiation of specific cellular immune responses. This review focuses on current evidence for HR-HPV manipulation of antigen presentation in dendritic cells and escape from host immunity

NKT cells inhibit antigen-specific effector CD8 T cell induction to skin viral proteins

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing pancreatic β cells. Therapies need to incorporate strategies to overcome the genetic defects that impair induction or maintenance of peripheral T-cell tolerance and contribute to disease development. We tested whether the enforced expression of an islet autoantigen in antigen-presenting cells (APC) counteracted peripheral T-cell tolerance defects in autoimmune-prone NOD mice. We observed that insulin-specific CD8(+) T cells transferred to mice in which proinsulin was transgenically expressed in APCs underwent several rounds of division and the majority were deleted. Residual insulin-specific CD8(+) T cells were rendered unresponsive and this was associated with TCR downregulation, loss of tetramer binding and expression of a range of co-inhibitory molecules. Notably, accumulation and effector differentiation of insulin-specific CD8(+) T cells in pancreatic lymph nodes was prominent in non-transgenic recipients but blocked by transgenic proinsulin expression. This shift from T-cell priming to T-cell tolerance exemplifies the tolerogenic capacity of autoantigen expression by APC and the capacity to overcome genetic tolerance defects

Langerhans cell homeostasis and activation is altered in hyperplastic human papillomavirus type 16 E7 expressing epidermis

It has previously been shown that expression of human papillomavirus type 16 (HPV) E7 in epidermis causes hyperplasia and chronic inflammation, characteristics of pre-malignant lesions. Importantly, E7-expressing epidermis is strongly immune suppressed and is not rejected when transplanted onto immune competent mice. Professional antigen presenting cells are considered essential for initiation of the adaptive immune response that results in graft rejection. Langerhans cells (LC) are the only antigen presenting cells located in normal epidermis and altered phenotype and function of these cells may contribute to the immune suppressive microenvironment. Here, we show that LC are atypically activated as a direct result of E7 expression in the epidermis, and independent of the presence of lymphocytes. The number of LC was significantly increased and the LC are functionally impaired, both in migration and in antigen uptake. However when the LC were extracted from K14E7 skin and matured in vitro they were functionally competent to present and cross-present antigen, and to activate T cells. The ability of the LC to present and cross-present antigen following maturation supports retention of full functional capacity when removed from the hyperplastic skin microenvironment. As such, opportunities are afforded for the development of therapies to restore normal LC function in hyperplastic skin

Recruitment of Antigen Presenting Cells to Skin Draining Lymph Node From HPV16E7-Expressing Skin Requires E7-Rb Interaction.

"High-risk" human papillomaviruses (HPV) infect keratinocytes of squamous epithelia. The HPV16E7 protein induces epithelial hyperplasia by binding Rb family proteins and disrupting cell cycle termination. Murine skin expressing HPV16E7 as a transgene from a keratin 14 promoter (K14.E7) demonstrates epithelial hyperplasia, dysfunctional antigen presenting cells, ineffective antigen presentation by keratinocytes, and production of immunoregulatory cytokines. Furthermore, grafted K14.E7 skin is not rejected from immunocompetent non-transgenic recipient animals. To establish the contributions of E7, of E7-Rb interaction and of epithelial hyperplasia to altered local skin immunity, K14.E7 skin was compared with skin from K14.E7 mice heterozygous for a mutant Rb unable to bind E7 (K14.E7xRbΔL/ΔL mice), that have normoplastic epithelium. Previously, we demonstrated that E7-speicfic T cells do not accumulate in K14.E7xRbΔL/ΔL skin grafts. Here, we further show that K14.E7xRbΔL/ΔL skin, like K14.E7 skin, is not rejected by immunocompetent non-transgenic animals. There were fewer CD11b+ antigen presenting cells in skin draining lymph nodes from animals recipient of K14.E7xRbΔL/ΔL grafts, when compared with animals receiving K14.E7 grafts or K5mOVA grafts. Maturation of migratory DCs derived from K14.E7xRbΔL/ΔL grafts found in the draining lymph nodes is significantly lower than that of K14.E7 grafts. Surprisingly, K14.E7xRbΔL/ΔL keratinocytes, unlike K14.E7 keratinocytes, are susceptible to E7 directed CTL-mediated lysis in vitro. We conclude that E7-Rb interaction and its associated epithelial hyperplasia partially contribute to the suppressive local immune responses in area affected by HPV16E7 expression

Inactivation of human immunodeficiency virus (HIV)-1 envelope-specific CD8+ cytotoxic T lymphocytes by free antigenic peptide: A self-veto mechanism?

Free peptide has been found to inhibit cytotoxic T lymphocyte (CTL) activity, and veto cells bearing peptide-major histocompatibility complex (MHC) complexes have been found to inactivate CTL, but the two phenomenon have not been connected. Here we show that a common mechanism may apply to both CD8+ CTL lines or clones specific for a determinant of the human immunodeficiency virus (HIV)-1 IIIB envelope protein gp160, P18IIIB, are inhibited by as little as 10 min exposure to the minimal 10-mer peptide, 1- 10, within P18IIIB, free in solution, in contrast to peptide already bound to antigen-presenting cells (APC), which does not inhibit. Several lines of evidence suggest that the peptide must be processed and presented by H-2D(d) on the CTL itself to the specific T cell receptor (TCR) to be inhibitory. The inhibition was not killing, in that CTL, did not kill 51Cr-labeled sister CTL in the presence of free peptide, and in missing experiments with CTL lines of different specificities restricted by the same MHC molecule, D(d), the presence of free peptide recognized by one CTL line did not inhibit the activity of the other CTL, line that could present the peptide. Also, partial recovery of activity could be elicited by restimulation with cell-bound peptide, supporting the conclusion that neither fratricide nor suicide (apoptosis) was involved. The classic veto phenomenon was ruled out by failure of peptide-bearing CTL to inactivate others. Using pairs of CTL lines of differing specificity but similar MHC restriction, each pulsed with the peptide for which the other is specific, we showed that the minimal requirement is simultaneous engagement of the TCR and class I MHC molecules of the same cell. This could occur in single cells or pairs of cells presenting peptide to each other. Thus, mechanistically the inhibition is analogous to veto, and might be called self-veto. As a clue to a possible mechanism, we found that free 1-10 peptide induced apparent downregulation of expression of specific TCR as well as interleukin 2 receptor, CD69, lymphocyte function-associated antigen 1, and CD8. This self-veto effect also has implication for in vivo immunization and mechanisms of viral escape from CTL immunity

HPV16 E7-driven epithelial hyperplasia promotes impaired antigen presentation and regulatory T cell development

Human papillomaviruses (HPV) infect keratinocytes and can lead to hyperproliferative dysplasia and malignant transformation if not cleared by the immune system. HPV has evolved an array of mechanisms to evade and manipulate the immune system to improve replication efficiency and promote persistent infection. We here demonstrate that hyperproliferative skin expressing the high-risk HPV16 E7 oncogene as a transgene drives immune-modulation of dendritic cells (DCs) resulting in reduced capacity to take up antigen and prime effector CD4 T cell responses. The phenotype of DCs in the E7-expressing hyperproliferative skin was not reversible by activation through intradermal immunization. Naïve CD4 T cells primed by E7-driven hyperproliferative skin acquired FoxP3 expression and an anergic phenotype. DC and T help modulation was dependent on E7-Rb interaction-driven epithelial hyperproliferation, rather than on expression of E7, as inhibition of binding of E7 to retinoblastoma protein, and of consequent epithelial hyperplasia was associated with normal skin DC phenotype, and Th1 effector responses to immunization were restored. We conclude that HPV-induced epithelial hyperplasia modulates epithelial DCs and inhibits Th1 immunity while polarizing T cell differentiation to a regulatory or anergic phenotype

Evaluation of techniques for extraction of hordein and glutelin from barley seed and a comparison of protein composition of Bomi and RisØ 1508

Whole seed of barley (cv. Julia) was ground, and the meal extracted to remove lipids, non-protein nitrogen compounds, albumins, and globulins. Four procedures for extracting hordein and glutelin from this meal were then compared. The composition of the isolated fractions was monitored by amino acid analysis and SDS polyacrylamide gel electrophoresis. More hordein was extracted by 55% (v/v) propan-2-ol containing 2% (v/v) 2-mercaptoethanol at 60 °C than at 20 °C or by sequential extraction with 55% propan-2-ol alone followed by 55% propan-2-ol plus 0.6% 2-mercaptoethanol. After hordein extraction glutelins were successfully extracted from the residual meal by reduction and alkylation in buffer containing 8 M urea, and were precipitated by dialysis against water. Small amounts of hordein were recovered from the alkylated glutelin by washing with hot 70% (v/v) ethanol plus 0.7% (v/v) acetic acid. The acid alcohol-insoluble glutelin was free from hordein polypeptides. Glutelins were also extracted sequentially using borate buffer at pH 10 with 0.6% mercaptoethanol followed by the same buffer with 1% SDS. Two procedures were used to compare the hordein and glutelin composition of endosperms of high lysine (RisØ 1508) and normal (Bomi, Julia) barley varieties. The hordein extracted at 60°C by 55% propan-2-ol plus 2% 2-mercaptoethanol represented almost 50% of the total N of the endosperm of Bomi and Julia, and 16% of RisØ 1508. The high lysine mutant (RisØ 1508) had more glutelin and salt-soluble nitrogen than Bomi. Electrophoretic analysis of the component polypeptides of the hordein of Bomi and RisØ 1508 showed several differences in the bands present, and in their relative proportions. In contrast the hordein-free glutelins of all three varieties appeared to have similar polypeptide compositions. Investigation of the salt-soluble fraction confirmed that the high lysine gene in RisØ 1508 results in increases in both protein and non-protein nitrogen components. The results obtained on the distribution of nitrogen between the various fractions in the seeds of Bomi and RisØ 1508 and on the amino acid analysis and polypeptide composition differ considerably from those published by other workers, in which a classical Osborne type extraction was used, and we conclude that such methods should not be used for barley