Specific CD8+ T cells recognize human herpesvirus 6B

The importance of human herpesvirus 6 (HHV‐6) species as human pathogens is increasingly appreciated. However, we do not understand how infection is controlled in healthy virus carriers, and why control fails in patients with disease. Other persistent viruses are under continuous surveillance by antigen‐specific T cells, and specific T‐cell repertoires have been well characterized for some of them. In contrast, knowledge on HHV‐6‐specific T‐cell responses is limited, and missing for CD8⁺ T cells. Here we identify CD8⁺ T‐cell responses to HHV‐6B, the most widespread HHV‐6 species, in healthy virus carriers. HHV‐6B‐specific CD8⁺ T‐cell lines and clones recognized HLA‐A2‐restricted peptides from the viral structural proteins U54 and U11, and displayed various antigen‐specific antiviral effector functions. These CD8⁺ T cells specifically recognized HHV‐6B‐infected primary CD4⁺ T cells in an HLA‐restricted manner, produced antiviral cytokines, and killed infected cells, whereas HHV‐6A‐infected cells were not recognized. Thus, HHV‐6B‐specific CD8⁺ T cells are likely to contribute to control of infection, overcoming the immunomodulatory effects exerted by the virus. Potentially, HHV‐6‐associated disease could be addressed by active or passive immunotherapy that reconstitutes virus‐specific CD8⁺ T‐cell responses.     

CTL recognition of HIV‐1‐infected cells via cross‐recognition of multiple overlapping peptides from a single 11‐mer Pol sequence

It is known that overlapping HIV‐1 peptides of different lengths can be presented by a given HLA class I molecule. However, the role of those peptides in CD8⁺ T cells recognition of HIV‐1‐infected cells remains unclear. Here we investigated the recognition of overlapping 8‐mer to 11‐mer peptides of Pol 155–165 by HLA‐B*54:01‐restricted CD8⁺ T cells. The analysis of ex vivo T cells using ELISPOT and tetramer binding assays showed that there were different patterns of CD8⁺ T‐cell responses to these peptides among chronically HIV‐1‐infected HLA‐B*54:01⁺ individuals, though the response to the 9‐mer peptide was the strongest among them. CD8⁺ T‐cell clones with TCRs specific for the 9‐mer, 10‐mer, and/or 11‐mer peptides effectively killed HIV‐1‐infected cells. Together, these results suggest that the 9‐mer and 10‐mer peptides could be predominantly presented by HLA‐B*54:01, though it remains possible that the 11‐mer peptide was also presented by this HLA allele. The present study demonstrates effective CD8⁺ T‐cell recognition of HIV‐1‐infected cells via presentation of multiple overlapping HIV‐1 peptides and cross‐recognition by the CD8⁺ T cells.     

Measles virus-induced suppression of immune responses

Summary: Measles is an important cause of child mortality that has a seemingly paradoxical interaction with the immune system. In most individuals, the immune response is successful in eventually clearing measles virus (MV) infection and in establishing life-long immunity. However, infection is also associated with persistence of viral RNA and several weeks of immune suppression, including loss of delayed type hypersensitivity responses and increased susceptibility to secondary infections. The initial T-cell response includes CD8⁺ and T-helper 1 CD4⁺ T cells important for control of infectious virus. As viral RNA persists, there is a shift to a T-helper 2 CD4⁺ T-cell response that likely promotes B-cell maturation and durable antibody responses but may suppress macrophage activation and T-helper 1 responses to new infections. Suppression of mitogen-induced lymphocyte proliferation can be induced by lymphocyte infection with MV or by lymphocyte exposure to a complex of the hemagglutinin and fusion surface glycoproteins without infection. Dendritic cells (DCs) are susceptible to infection and can transmit infection to lymphocytes. MV-infected DCs are unable to stimulate a mixed lymphocyte reaction and can induce lymphocyte unresponsiveness through expression of MV glycoproteins. Thus, multiple factors may contribute both to measles-induced immune suppression and to the establishment of durable protective immunity.     

Antitumor cytotoxic T-lymphocyte response in human lung carcinoma: identification of a tumor-associated antigen

Summary: We have isolated several cytotoxic T lymphocyte (CTL) clones from lymphocytes infiltrating a lung carcinoma of a patient with long survival. These clones showed a CD3⁺, CD8⁺, CD4^–, CD28^– phenotype and expressed a T‐cell receptor (TCR) encoded either by Vβ8‐Jβ1.5 or Vβ22‐Jβ1.4 rearrangements. Functional studies indicated that these clones mediated a high human leukocyte antigen (HLA)‐A2.1‐restricted cytotoxic activity against the autologous tumor cell line. Interestingly, TCRβ chain gene usage indicated that CTL clones identified in vitro were selectively expandedin vivo at the tumor site as compared to autologous peripheral blood lymphocytes (PBL). These findings provide evidence that an immune response may take place in non‐small cell lung carcinoma and that effector T cells may contribute to tumor regression. Further study indicated that the CTL clones recognized the same decamer peptide encoded by a mutated α‐actinin‐4 gene. Using tetramers of soluble HLA‐A2 molecules loaded with the mutated antigenic peptide, we have derived several anti‐α‐actinin‐4 T‐cell clones from patient PBL. These CTL, recognizing a truly tumor‐specific antigen, may play a role in the clinical evolution of this lung cancer patient. Adoptive transfer of CTL clones in a SCID/NOD mice model transplanted with autologous tumor supported their antitumor effect in vivo.     

Virus-specific cytotoxic T cell responses are associated with immunity of the cottontop tamarin to Epstein–Barr virus (EBV)

The cytotoxic responses of peripheral blood lymphocytes from cottontop tamarins to in vitro re-stimulation with autologous lymphoblastoid cell lines (LCL) were assayed. Lymphocytes from immune tamarins that had recovered from EBV challenge developed potent cytotoxicity for natural killer (NK) cell targets and for autologous LCL. The cytotoxicity for LCL targets was EBV-specific, as B cell blasts uninfected with EBV were not killed. The cell lines could be maintained by repeated stimulation with LCL and the addition of IL-2. Flow cytometry showed that they were T cell lines expressing CD2, CD3, CD4, CD8 and CD25. Dual-colour flow cytometry revealed two subpopulations, one CD4⁺ CD8⁺ population and the other CD4⁻ CD8⁺. After separation by magnetic cell sorting both subpopulations were shown to be cytotoxic and the CD4⁺ CD8⁺ fraction was also shown to be MHC class II-restricted; the MHC restriction of the CD8⁺ subpopulation could not be determined. The unseparated T cells and both the subpopulations were able to inhibit LCL outgrowth in vitro. In contrast, PBL from naive tamarins stimulated by autologous LCL developed less NK cell cytotoxicity and little cytotoxicity for LCL. The cytotoxic response was enhanced at higher levels of LCL stimulation, but the cells were unable to inhibit LCL outgrowth in vitro. We conclude that cytotoxic responses capable of inhibiting LCL growth in vitro correlate with in vivo immunity in the tamarin model and provide a basis for understanding the mechanism of vaccine-induced immune protection.     

T cell anergy and activation are associated with suboptimal humoral responses to measles revaccination in HIV‐infected children on anti‐retroviral therapy in Nairobi,Kenya

HIV‐infected children are less capable of mounting and maintaining protective humoral responses to vaccination against measles compared to HIV‐uninfected children. This poses a public health challenge in countries with high HIV burdens. Administration of anti‐retroviral therapy (ART) and revaccinating children against measles is one approach to increase measles immunity in HIV‐infected children, yet it is not effective in all cases. Immune anergy and activation during HIV infection are factors that could influence responses to measles revaccination. We utilized a flow cytometry‐based approach to examine whether T cell anergy and activation were associated with the maintenance of measles‐specific immunoglobulin (Ig)G antibodies generated in response to measles revaccination in a cohort of HIV‐infected children on ART in Nairobi, Kenya. Children who sustained measles‐specific IgG for at least 1 year after revaccination displayed significantly lower programmed cell death 1 (PD‐1) surface expression on CD8⁺ T cells on a per‐cell basis and exhibited less activated CD4⁺ T cells compared to those unable to maintain detectable measles‐specific antibodies. Children in both groups were similar in age and sex, CD4⁺ T cell frequency, duration of ART treatment and HIV viral load at enrolment. These data suggest that aberrant T cell anergy and activation are associated with the impaired ability to sustain an antibody response to measles revaccination in HIV‐infected children on ART.     

Human herpesvirus 6B immediate‐early I protein contains functional HLA‐A*02, HLA‐A*03, and HLA‐B*07 class I restricted CD8+ T‐cell epitopes

Human herpesvirus 6B (HHV‐6B) is a ubiquitous pathogen with frequent reactivation observed in immunocompromised patients such as BM transplant (BMT) recipients. Adoptive immunotherapy is a promising therapeutic avenue for the treatment of opportunistic infections, including herpesviruses. While T‐cell immunotherapy can successfully control CMV and EBV reactivations in BMT recipients, such therapy is not available for HHV‐6 infections, in part due to a lack of identified protective CD8⁺ T‐cell epitopes. Our goal was to identify CD8⁺ T‐cell viral epitopes derived from the HHV‐6B immediate‐early protein I and presented by common human leukocyte Ag (HLA) class I alleles including HLA‐A*02, HLA‐A*03, and HLA‐B*07. These epitopes were functionally tested for their ability to induce CD8⁺ T‐cell expansion and kill HHV‐6‐infected autologous cells. Cross‐reactivity of specific HHV‐6B‐expanded T cells against HHV‐6A‐infected cells was also confirmed for a conserved epitope presented by HLA‐A*02 molecule. Our findings will help push forward the field of adoptive immunotherapy for the treatment and/or the prevention of HHV‐6 reactivation in BMT recipients.     

Lysis of CD4+ lymphocytes by non-HLA-restricted cytotoxic T lymphoe from HIV-infected individuals

Individuals infected with HIV have elevated numbers of total and activated CD8⁺ lymphocytes in peripheral blood. CD8⁺ lymphocytes from HIV-infected individuals have been shown to mediate non-human histocompatibility-linked antigen (HLA)-restricted suppression of viral replication, HLA-restricted killing of cells expressing HIV antigens, and killing of uninfected lymphocytes. We studied CD8⁽ T lymphocytes that lysed autologous CD4+ lymphocytes, hetcrologous CD4¹ lymphocytes from HIV-infected individuals and uninfected CD4⁺ lymphocytes. Killing in all cases required T cell receptor (TCR)-mediated recognition or triggering. However, these CD8 cytotoxic T lymphocytes (CTL) killed HLA class I mismatched CD4* lymphocytes and CD4⁴ lymphocytes treated with a MoAb against HLA-A, B and C antigens (PA2.6) which blocks HLA class I-restricted killing. HLA class H-negativc CD4* T lymphoma cells (CEM.NK^R) were also killed by anti-CD3 inhibited CTL. Stimulation of peripheral blood lymphocytes (PBL) from HIV-infected individuals, but not uninfected controls, with concanavalin A (Con A) and IL-2, induced non-HLA-restricted TCR aft¹, CD8^f CTL which lysed CD4⁺ lymphocytes. Activation ofCD4’lymphocytes increased their susceptibility to CD8^f CTL-mediated lysis. In HIV infection, a population of non-HLA-restricted CTL which lyse activated CD4⁺ lymphocytes is expanded. The expansion of CTL with unusual characteristics is interesting, because the stimulus for this expansion is unknown. CTL which recognize activated CD4⁺ cells could play a role in immune regulation and the pathogenesis of A IDS.     

Effector CD8+T cells are suppressed by measles virus infection during delayed type hypersensitivity reaction

Measles virus infection reduces or abolishes delayed type hypersensitivity reactions (DTH) in humans. We have previously shown that the primary 2,4-dinitrofluorobenzene (DNFB) response is temporarily suppressed by measles virus in cotton rats. Here, we demonstrate that also the secondary DNFB response (cutaneous hypersensitivity [CHS]) is suppressed in cotton rats by measles virus infection. As in mice, DNFB specific CD8 T cells are the predominant T cell response in cotton rats. After MV infection, CD8 T cells are reduced in their proliferative capacity whereas the CD4/CD8 ratio, the number and activation status of CD8 T cells is not affected. As a result of impaired proliferation of DNFB specific T cells the DTH response (measured as ear swelling) is reduced in measles virus infected cotton rats. At the same time as DNFB specific T cell responses are suppressed, spontaneous proliferation of lymphocytes as evidence for immune activation is found.     

The impact of HLA class I and EBV latency‐II antigen‐specific CD8+ T cells on the pathogenesis of EBV+ Hodgkin lymphoma

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein–Barr virus (EBV) latency‐II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV⁺cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA‐A*02 is protective in EBV⁺cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA‐A*02^– versus HLA‐A*02⁺ EBV⁺cHL patients, suggesting that LMP2A‐specific CD8⁺ T cell anti‐tumoral immunity may be relatively ineffective in HLA‐A*02^– EBV⁺cHL. To ascertain the impact of HLA class I on EBV latency antigen‐specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV⁺cHL, the magnitude of ex‐vivo LMP1/2A‐specific CD8⁺ T cell responses was elevated in HLA‐A*02⁺ patients. Furthermore, in a controlled in‐vitro assay, LMP2A‐specific CD8⁺ T cells from healthy HLA‐A*02 heterozygotes expanded to a greater extent with HLA‐A*02‐restricted compared to non‐HLA‐A*02‐restricted cell lines. In an extensive analysis of HLA class I‐restricted immunity, immunodominant EBNA3A/3B/3C‐specific CD8⁺ T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A‐specific responses were confined largely to HLA‐A*02. Our results demonstrate that HLA‐A*02 mediates a modest, but none the less stronger, EBV‐specific CD8⁺ T cell response than non‐HLA‐A*02 alleles, an effect confined to EBV latency‐II antigens. Thus, the protective effect of HLA‐A*02 against EBV⁺cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency‐II antigen‐specific CD8⁺ T cell hierarchies.     

Distinct Pattern of Immunophenotypic Features of Innate and Adaptive Immunity as a Putative Signature of Clinical and Laboratorial Status of Patients with Localized Cutaneous Leishmaniasis

In this study, we have analysed the phenotypic features of innate/adaptive immunity of patients with localized cutaneous leishmaniasis (LCL), categorized according to their clinical/laboratorial status, including number of lesion (L1; L2–4), days of illness duration (≤60;>60) and positivity in the Montenegro skin test (MT^?;MT⁺). Our findings highlighted a range of phenotypic features observed in patients with LCL (↑%HLA‐DR⁺ neutrophils; ↑CD8⁺ HLA‐DR⁺/CD4⁺ HLA‐DR⁺ T cell ratio; ↑HLA‐DR in B lymphocytes, ↑%CD23⁺ neutrophils, monocytes and B cells; ↑α‐Leishmania IgG and ↑serum + ). Selective changes were observed in L1 (↑%HLA‐DR⁺ neutrophils, ↑CD8⁺ HLA‐DR⁺/CD4⁺ HLA‐DR⁺ T cell ratio and ↑serum + ) as compared to L2–4 (↑%CD5^? B cells; ↑CD23⁺ B cells and ↑α‐Leishmania IgG). Whilst ≤60 presented a mixed profile of innate/adaptive immunity (↓%CD28⁺ neutrophils and ↑%CD4⁺ T cells), >60 showed a well‐known leishmanicidal events (↑CD8⁺ T cells; ↑serum + and ↑α‐Leishmania IgG). MT⁺ patients showed increased putative leishmanicidal capacity (↑%HLA‐DR⁺ neutrophils; ↑%CD23⁺ monocytes; ↑CD8⁺ HLA‐DR⁺/CD4⁺ HLA‐DR⁺ T cell ratio and ↑ serum + ). Overall, a range of immunological biomarkers illustrates the complex immunological network associated with distinct clinical/laboratorial features of LCL with applicability in clinical studies.     

HLA class II-restricted recognition of common tumor epitopes on human melanoma cells by CD4+ melanoma-infiltrating lymphocytes

CD4⁺ T cell clones derived from lymphocytes infiltrating four human melanomas specifically recognized melanoma-derived tumor epitopes as shown by secretion of tumor necrosis factor (TNF) in vitro upon interaction with autologous melanoma cells, whereas they did not recognize HLA class II-expressing autologous lymphoblasts or HLA class II mismatched allogeneic melanoma cells. Specificity was further established by demonstrating that TNF responses to tumor cells were inhibited by HLA-DR or HLA-DQ monoclonal antibodies. Most of these clones cross-reacted with allogeneic melanoma cells expressing a potentially restricting HLA allele or a structurally similar one. These data show that shared epitopes of human melanoma cells presented on HLA class II molecules are frequently recognized by autologous CD4⁺ T lymphocytes.     

The differential impact of natural killer (NK) cell education via KIR2DL3 and KIR3DL1 on CCL4 secretion in the context of in‐vitro HIV infection

Carriage of certain inhibitory natural killer (NK) cell receptor (iNKR)/HLA ligand pairs is associated with protection from infection and slow time to AIDS implicating NK cells in HIV control. NK cells acquire functional potential through education, which requires the engagement of iNKRs by their human leucocyte antigen (HLA) ligands. HIV infection down‐regulates cell surface HLA‐A/B, but not HLA‐C/E. We investigated how NK cell populations expressing combinations of the iNKRs NKG2A, KIR2DL3 (2DL3) and KIR3DL1 (3DL1) responded to autologous HIV infected CD4 (iCD4) cells. Purified NK cells from HIV‐uninfected individuals were stimulated with autologous HIV iCD4 or uninfected CD4 T cells. Using flow cytometry we gated on each of the 8 NKG2A^+/–2DL3^+/–3DL1^+/‐ populations and analysed all possible combinations of interferon (IFN)‐γ, CCL4 and CD107a functional subsets responding to iCD4 cells. Infected CD4 cells induced differential frequencies of NKG2A^+/–2DL3^+/–3DL1^+/– populations with total IFN‐γ⁺, CCL4⁺ and CD107a⁺ functional profiles. 2DL3⁺NKG2A⁺ NK cells had a higher frequency of responses to iCD4 than other populations studied. A higher frequency of 2DL3⁺ NK cells responded to iCD4 from individuals that were not HLA‐C1 homozygotes. These results show that 2DL3⁺ NK cells are mediators of HIV‐specific responses. Furthermore, responses of NK cell populations to iCD4 are influenced not only by NK cell education through specific KIR/HLA pairs, but also by differential HIV‐mediated changes in HLA expression.     

Distinct activation phenotype of a highly conserved novel HLA‐B57‐restricted epitope during dengue virus infection

Variation in the sequence of T‐cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T‐cell responses during second heterologous infections. We identified a highly conserved, novel, HLA‐B57‐restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57‐NS1_26–34‐specific CD8⁺ T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer‐positive T‐cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57‐NS1_26–34‐specific and other DENV epitope‐specific CD8⁺ T cells, as well as total CD8⁺ T cells, expressed an activated phenotype (CD69⁺ and/or CD38⁺) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope‐specific CD8⁺ T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen‐specific T‐cell activation.     

Analysis of nucleophosmin–anaplastic lymphoma kinase (NPM‐ALK)‐reactive CD8+ T cell responses in children with NPM‐ALK+ anaplastic large cell lymphoma

Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK‐positive anaplastic large cell lymphoma (ALCL) have been detected using peptide‐based approaches in individuals preselected for human leucocyte antigen (HLA)‐A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)‐ALK‐specific CD8⁺ T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK‐specific CD8⁺ T cells. Autologous dendritic cells (DCs) transfected with in‐vitro‐transcribed RNA (IVT‐RNA) encoding NPM–ALK were used as antigen‐presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon‐gamma enzyme‐linked immunospot (ELISPOT) assays with NPM–ALK‐transfected autologous DCs as well as CV‐1 in Origin with SV40 genes (COS‐7) cells co‐transfected with genes encoding the patients’ HLA class I alleles and with NPM–ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM–ALK‐specific CD8⁺ T cell responses were detected in three of five ALK‐positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti‐ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM–ALK‐specific CD8⁺ T cell responses were restricted by HLA‐C‐alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM–ALK‐reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.     

Intrathecal CD4+ and CD8+ T‐cell responses to endogenously synthesized candidate disease‐associated human autoantigens in multiple sclerosis patients

MS pathology is potentially orchestrated by autoreactive T cells, but the antigens recognized remain unknown. A novel APC/T‐cell platform was developed to determine intrathecal CD4⁺ and CD8⁺ T‐cell responses to candidate MS‐associated autoantigens (cMSAg) in clinically isolated syndrome (CIS, n = 7) and MS (n = 6) patients. Human cMSAg encoding open reading frames (n = 8) were cloned into an Epstein–Barr virus (EBV)‐based vector to express cMSAg at high levels in EBV‐transformed B‐cells (BLCLs). Human cMSAg cloned were myelin‐associated and ‐oligodendrocyte glycoprotein, myelin basic protein, proteolipid protein, ATP‐dependent potassium channel ATP‐dependent inwards rectifying potassium channel 4.1, S100 calcium‐binding protein B, contactin‐2, and neurofascin. Transduced BLCLs were used as autologous APC in functional T‐cell assays to determine cMSAg‐specific T‐cell frequencies in cerebrospinal fluid derived T‐cell lines (CSF‐TCLs) by intracellular IFN‐γ flow cytometry. Whereas all CSF‐TCL responded strongly to mitogenic stimulation, no substantial T‐cell reactivity to cMSAg was observed. Contrastingly, measles virus fusion protein‐specific CD4⁺ and CD8⁺ T‐cell clones, used as control of the APC/T‐cell platform, efficiently recognized transduced BLCL expressing their cognate antigen. The inability to detect substantial T‐cell reactivity to eight human endogenously synthesized cMSAg in autologous APC do not support their role as prominent intrathecal T‐cell target antigens in CIS and MS patients early after onset of disease.     

Selective antigen‐specific CD4+ T‐cell,but not CD8+ T‐ or B‐cell,tolerance corrupts cancer immunotherapy

Self‐tolerance, presumably through lineage‐unbiased elimination of self‐antigen‐specific lymphocytes (CD4⁺ T, CD8⁺ T, and B cells), creates a formidable barrier to cancer immunotherapy. In contrast to this prevailing paradigm, we demonstrate that for some antigens, self‐tolerance reflects selective elimination of antigen‐specific CD4⁺ T cells, but preservation of CD8⁺ T‐ and B‐cell populations. In mice, antigen‐specific CD4⁺ T‐cell tolerance restricted CD8⁺ T‐ and B‐cell responses targeting the endogenous self‐antigen guanylyl cyclase c (GUCY2C) in colorectal cancer. Although selective CD4⁺ T‐cell tolerance blocked GUCY2C‐specific antitumor immunity and memory responses, it offered a unique solution to the inefficacy of GUCY2C vaccines through recruitment of self‐antigen‐independent CD4⁺ T‐cell help. Incorporating CD4⁺ T‐cell epitopes from foreign antigens into vaccines against GUCY2C reconstituted CD4⁺ T‐cell help, revealing the latent functional capacity of GUCY2C‐specific CD8⁺ T‐ and B‐cell pools, producing durable antitumor immunity without autoimmunity. Incorporating CD4⁺ T‐cell epitopes from foreign antigens into vaccines targeting self‐antigens in melanoma (Trp2) and breast cancer (Her2) produced similar results, suggesting selective CD4⁺ T‐cell tolerance underlies ineffective vaccination against many cancer antigens. Thus, identification of self‐antigens characterized by selective CD4⁺ T‐cell tolerance and abrogation of such tolerance through self‐antigen‐independent T‐cell help is essential for future immunotherapeutics.     

CD8high(CD57+) T cells in normal,healthy individuals specifically suppress the generation of cytotoxic T lymphocytes to Epstein-Barr virus-transformed B cell lines

We have previously identified two subsets of CD8⁺, CD57⁺ lymphocytes in normal peripheral blood: i) T cells expressing high levels [CD8^high(CD57⁺)] and ii) natural killer cells expressing low levels of surface CD8 [CD8^low(CD57⁺)]. We investigated the cytotoxic and suppressive function of CD8^high(CD57⁺) T lymphocytes from normal, healthy individuals using standard chromium-release assays and limiting dilution analysis. In normal, healthy subjects, this cell subset suppressed the generation of cytotoxic T lymphocytes (CTL) to autologous, Epstein-Barr virus (EBV)-transformed B cell lines (BCL). Depletion of CD8^high(CD57⁺) T lymphocytes from peripheral blood mononuclear cells (PBMC) resulted in a three- to sevenfold rise in CTL precursor frequency to autologous EBV-transformed BCL, but not allogeneic PBMC or BCL by LDA. Replacement of CD8^high(CD57⁺) T lymphocytes in limiting dilution cultures led to the dose-dependent suppression of EBV-specific, but not allogeneic, CTL generation. Supernatant from CD8^high(CD57⁺) T lymphocytes cultured with autologous BCL did not exhibit suppression, suggesting that soluble factors were not responsible. As CD8^high(CD57⁺) T lymphocytes did not, themselves, exhibit cytotoxicity against autologous BCL, removal of BCL stimulator cells in co-culture was not the mechanism of suppression. Furthermore, while the CD8^high(CD57⁺) T lymphocytes from healthy subjects suppressed the generation of CTL to autologous BCL, they did not suppress the cytotoxic activity of established mixed lymphocyte reactions or peptide-specific CTL clones, as has been reported in bone marrow transplant recipients and human immunodeficiency virus patients. This suggests that CD8^high(CD57⁺) T lymphocytes from healthy subjects suppress the generation of, rather than killing by, CTL in a contact-dependent manner. To our knowledge, this is the first identification of a phenotypically distinct subset of human CD8⁺ T cells that can suppress generation of antigen-specific major histocompatibility complex class I-restricted CTL.     

HLA-DR and HLA-DP Restricted Epitopes from Human Cytomegalovirus Glycoprotein B Recognized by CD4+ T-Cell Clones from Chronically Infected Individuals

Purpose

Helper CD4⁺ T cells presumably play a major role in controlling cytomegalovirus (CMV) by providing help to specific B and CD8⁺ cytotoxic T cells, as well as through cytotoxicity-mediated mechanisms. Since CMV glycoprotein B (gB) is a major candidate for a subunit vaccine against CMV, we searched for gB-epitopes presented by human leukocyte antigen (HLA)-class II molecules.

Methods

Dendritic cells obtained from CMV-seropositive donors were loaded with a recombinant gB and co-cultured with autologous CD4⁺ T cells. Microcultures that specifically recognized gB were cloned by limiting dilution using autologous Epstein-Barr virus (EBV)-immortalized B cells pulsed with gB as antigen-presenting cells. To pinpoint precisely the region encoding the natural epitope recognized by a given CD4⁺ clone, we assessed the recognition of recombinant Escherichia coli expressing gB-overlapping polypeptides after their processing by autologous EBV-B cells.

Results

We isolated several gB-specific CD4⁺ T-cell clones directed against peptides gB_190-204, gB_396-410, gB_22-36 and gB_598-617 presented by HLA-DR7, HLA-DP10 and HLA-DP2. While their precise role in controlling CMV infection remains to be established, gB-specific CD4⁺ T cells are likely to act by directly targeting infected HLA-class II cells in vivo, as suggested by their recognition of EBV-B cells infected by the Towne CMV strain.

Conclusions

The characterization of such gB-epitopes presented by HLA-class II should help to understand the contribution of CD4⁺ T-cell responses to CMV and may be of importance both in designing a vaccine against CMV infection and in immunomonitoring of subjects immunized with recombinant gB or with vectors encoding gB.     

Involvement of CD8+ T cells in protective immunity against murine blood‐stage infection with Plasmodium yoelii 17XL strain

When developing malaria vaccines, the most crucial step is to elucidate the mechanisms involved in protective immunity against the parasites. We found that CD8⁺ T cells contribute to protective immunity against infection with blood‐stage parasites of Plasmodium yoelii. Infection of C57BL/6 mice with P. yoelii 17XL was lethal, while all mice infected with a low‐virulence strain of the parasite 17XNL acquired complete resistance against re‐infection with P. yoelii 17XL. However, the host mice transferred with CD8⁺ T cells from mice primed only with P. yoelii 17XNL failed to acquire protective immunity. On the other hand, the irradiated host mice were completely resistant to P. yoelii 17XL infection, showing no grade of parasitemia when adoptively transferred with CD8⁺ T cells from immune mice that survived infection with both P. yoelii XNL and, subsequently, P. yoelii 17XL. These protective CD8⁺ T cells from immune WT mice had the potential to generate IFN‐γ, perforin (PFN) and granzyme B. When mice deficient in IFN‐γ were used as donor mice for CD8⁺ T cells, protective immunity in the host mice was fully abrogated, and the immunity was profoundly attenuated in PFN‐deficient mice. Thus, CD8⁺ T cells producing IFN‐γ and PFN appear to be involved in protective immunity against infection with blood‐stage malaria.