Th2-type CD4(+) cells neither enhance nor suppress antitumor CTL activity in a mouse tumor model

Many cervical cancers express the E7 protein of human papillomavirus 16 as a tumor-specific Ag (TSA). To establish the role of E7-specific T cell help in CD8(+) CTL-mediated tumor regression, C57BL/6J mice were immunized with E7 protein or with a peptide (GF001) comprising a minimal CTL epitope of E7, together with different adjuvants, Immunized mice were challenged with an E7-expressing tumor cell line, EL4.E7. Growth of EL4.E7 was reduced following immunization with E7 and Quil-A (an adjuvant that induced a Th1-type response to E7) or with GF001 and Quil-A, Depletion of CD8(+) cells, but not CD4(+) cells, from an immunized animal abrogated protection, confirming that E7-specific CTL are necessary and sufficient for TSA-specific protection in this model. Immunization with E7 and Algammulin (an alum-based adjuvant) induced a Th2-like response and provided; no tumor protection. To investigate whether a Th2 T helper response to E7 could prevent the development of an E7-specific CTL-mediated protection, mice were simultaneously immunized with E7/Algammulin and GF001/Quil-A or, alternatively, were immunized with GF011/Quil-A 8 wk after immunization with E7/Algammulin, Tumor protection was observed in each case. We conclude that an established Th2 response to a TSA does not prevent the development of TSA-specific tumor protective CTL.

Both CD4(+) and CD8(+) lymphocytes reduce the severity of tissue lesions in murine systemic candidiasis, and CD4(+) cells also demonstrate strain-specific immunopathological effects

The role of T lymphocytes in host responses to sublethal systemic infection with Candida albicans was evaluated by mAb depletion of CD4(+) and CD8(+) cells from BALB/c and CBA/CaH mice, which develop mild and severe tissue damage, respectively. Depletion of CD4(+) lymphocytes from BALB/c mice markedly increased tissue damage, but did not alter the course of infection. In CBA/CaH mice, depletion of CD4+ cells abrogated tissue destruction in both brain and kidney at day 4 after infection, and significantly decreased fungal colonization in the brain. However, the severity of tissue lesions increased relative to controls from day 8 onwards. A small increase in tissue damage was evident in both mouse strains after depletion of CD8(+) cells. There were no major differences between days 4 end 8 after infection in cDNA cytokine profiles of CD4(+) lymphocytes from either BALB/c or CBA/CaH mice. After passive transfer into infected syngeneic recipients, spleen cells from infected CBA/CaH mice markedly increased tissue damage when compared to controls, and also caused a significant increase in fungal colonization in the brain. A similar transfer in BALB/c mice increased the number of inflammatory cells in and around the lesions, but had no effect on the fungal burden in brain and kidney. The data demonstrate that both CD4(+) and CD8(+) lymphocytes contribute to the reduction of tissue damage after systemic infection with C. albicans, and that the development and expression of CD4(+) lymphocyte effector function is influenced by the genetic background of the mouse.

Gene transfer and expression of a non-viral polycation-based vector in CD4(+) cells

CD4-selective targeting of an antibody-polycation-DNA complex was investigated The complex was synthesized with the anti-CD4 monoclonal antibody B-F5, polylysine(268) (pLL) and either the pGL3 control vector containing the luciferase reporter gene or the pGeneGrip vector containing the green fluorescent protein (GFP) gene. B-F5-pLL-DNA complexes inhibited the binding of I-125-B-F5 to CD4(+) Jurkat cells, while complexes synthesised either without B-F5 or using a non-specific mouse IgG1 antibody had little or no effect Expression of the luciferase reporter gene was achieved in Jurkat cells using the B-F5-pLL-pGL3 complex and was enhanced in the presence of PMA. Negligible luciferase activity was defected with the non-specific antibody complex in Jurkat cells or with the B-F5-pLL-pGL3 complex in the CD4(-) K-562 cells. Using complexes synthesised with the pGeneGrip vector, the transfection efficiency in Jurkat and K-562 cells was examined using confocal microscopy. More than 95% of Jurkat cells expressed GFP and the level of this expression was markedly enhanced by PMA. Negligible GFP expression was seen in K-562 cells or when B-F5 was replaced by a nonspecific antibody. Using flow cytometry, fluorescein-labelled complex showed specific targeting to CD4(+) cells in a mixed cell population from human peripheral blood. These studies demonstrate the selective transfection of CD4(+) T-lymphoid cells using a polycation-based gene delivery system. The complex may provide a means of delivering anti-HIV gene therapies to CD4(+) cells in vivo.

Aspects of the biology of the yabby Trypea australiensis (Dana) (Decapoda : Thalassinidea) and the potential of burrow counts as an indirect measure of population density

The thalassinidean shrimp Trypea australiensis (the yabby) commonly occurs on intertidal sandflats and subtidal regions of sheltered embayments and estuaries along the east coast of Australia and is harvested commercially and recreationally for use as bait by anglers. The potential for counts of burrow openings to provide a reliable indirect estimate of the abundance of yabbies was examined on intertidal sandflats on North Stradbroke Island (Queensland, Australia). The relationship between the number of burrow openings and the abundance of yabbies was generally poor and also varied significantly through time, casting doubt on previous estimates of abundance for this species based on unvalidated hole counts. Spatial and temporal variation in population density, the size at maturity and the reproductive period of the yabby were also assessed. Except for an initial peak in abundance as a result of recruitment, the density of yabbies was constant throughout the study but considerably less than that estimated from a previous study in the same area. Ovigerous females were recorded at 3 mm carapace length (CL) which is smaller than previously recorded for this species and thalassinideans in general. Small ovigerous females were found throughout the study, including the summer months, which is unusual for thalassinideans in the intertidal zone. It was hypothesised that in the intertidal zone, small female yabbies may be able to balance the metabolic demands of reproduction and respiration at higher temperatures than can larger females allowing them to reproduce in the warmer months.

Association of increased levels of heavy-chain ferritin with increased CD4(+) CD25(+) regulatory T-Cell levels in patients with melanoma

We have shown previously that melanoma cells in culture release heavy-chain ferritin (H-Ferritin) into supernatants and that this is responsible for the suppression of responses of peripheral blood lymphocytes stimulated by anti-CD3. These effects were mediated by activation of regulatory T cells to produce interleukin (IL)-10. In the present study, we examined whether a similar relation might exist between levels of H-Ferritin and activation of regulatory T cells in patients with melanoma. Ferritin levels were evaluated by ELISA and regulatory T-cell numbers were assessed by three-color flow cytometry to identify CD4(+) CD25(+) CD69(-) T cells. CD69 positive cells were excluded to avoid inclusion of normal activated CD4, CD25 expressing T cells. Measurements of H- and light-chain (L)-Ferritin by ELISA revealed that H- but not L-Ferritin was elevated in the circulation of melanoma patients. In addition, these studies revealed a marked increase in the number of CD4+ CD25+ CD69- T cells in such patients, compared with age-matched controls. The ratio of H-Ferritin:L-Ferritin correlated with the levels of regulatory T cells consistent with a causal relation between unbound H-Ferritin levels and the activation of regulatory T cells. H-Ferritin or regulatory T cells did not, however, correlate with the stage of the melanoma. These results provide evidence for the importance of H-Ferritin in the induction of regulatory T cells in patients with melanoma and provide additional insight into the suppression of immune responses in such patients.

Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection

Because CD4(+) T cells play a key role in aiding cellular immune responses, we wanted to assess whether increasing numbers of gene-engineered antigen-restricted CD4(+) T cells could enhance an antitumor response mediated by similarly gene-engineered CD8(+) T cells. In this study, we have used retroviral transduction to generate erbB2-reactive mouse T-cell populations composed of various proportions of CD4(+) and CD8(+) cells and then determined the antitumor reactivity of these mixtures. Gene-modified CD4(+) and CD8(+) T cells were shown to specifically secrete Tc1 (T cytotoxic-1) or Tc2 cytokines, proliferate, and lyse erbB2(+) tumor targets following antigen ligation in vitro. In adoptive transfer experiments using severe combined immunodeficient (scid) mice, we demonstrated that injection of equivalent numbers of antigen-specific engineered CD8(+) and CD4(+) T cells led to significant improvement in survival of mice bearing established lung metastases compared with transfer of unfractionated (largely CD8(+)) engineered T cells. Transferred CD4(+) T cells had to be antigen-specific (not just activated) and secrete interferon gamma (IFN-gamma) to potentiate the antitumor effect. Importantly, antitumor responses in these mice correlated with localization and persistence of gene-engineered T cells at the tumor site. Strikingly, mice that survived primary tumor challenge could reject a subsequent re-challenge. Overall, this study has highlighted the therapeutic potential of using combined transfer of antigen-specific gene-modified CD8(+) and CD4(+) T cells to significantly enhance T-cell adoptive transfer strategies for cancer therapy.

The role of CD4 T help in multiple vaccinations when using minimal CD8 T cell epitope peptides

Background: The fact that some cancers and viral infections can be controlled by effector CD8 T cells led to the possibility of utilising minimal CD8 T cell epitope peptides as vaccines. However using minimal CD8 T cell epitope peptide immunisations and a tumour protection model in mice, we have previously shown that functional memory CD8 T cells are not generated unless CD4 T help is provided at the time of CD8 T cell priming. Short-lived effector cells nevertheless are generated in the absence of T help. Aim: To determine the role of CD4 T help in multiple immunisations. Method: Minimal CD8 T cell peptides of HPV16 E7 protein and Ovalbumin were used (with adjuvants Quil-A or IFA) as immunogens in C57BL mice. The presence of effector CD8 T cells were determined by tumour protection assays and was quantified by IFN-gamma ELISPOT assays. Results: In the present study we show that unless T help is provided at the time CD8 T cells are primed, no CD8 effector cells are generated when boosted with the vaccine again in the absence of T help. Our results further show that this failure could be prevented by the inclusion of a T helper peptide during the primary or booster immunisations.

Cognate CD4(+) help elicited by resting DC does not impair CD8(+) T-cell tolerance induction

No abstract

A comparison of dissected follicle numbers and follicle counts on the ovarian surface for the evaluation of ovarian follicular populations in Bos indicus cows

Ovaries (n = 140) from 70 mixed-age multiparous, lactating Brahman cross (3/4-7/8 Bos indicus) cows were used to examine the hypothesis that counts of follicles visible on the surface of the ovaries of Bos indicus cows and their classification into diameter size classes, are closely correlated with numbers of follicles in those size classes found by complete dissection of the ovary. immediately after ovariectomy, mean diameters (long and short axes averaged) of all follicles greater than or equal to 2 mm visible on the surface of each ovary were measured. All follicles greater than or equal to 2 mm were dissected from the ovaries, excess stroma removed and follicle diameters measured under a stereomicroscope using an ocular graticule. For each ovary, follicles were classified in either small (8 mm) categories based on either diameters of surface or dissected follicles. Data for numbers of surface and dissected follicles (mean +/- SE) in small, medium, large categories and total follicle numbers, respectively, were 24.4 +/- 1.6 vs. 28.0 +/- 1.9, 1.6 +/- + 0.2 vs. 11.6 +/- 1.0, 0.5 +/- 0.1 vs. 0.7 +/- 0.1 and 26.4 +/- 1.6 vs. 40.4 +/- 2.5. Correlation coefficients (r) for counts of surface and dissected follicles in small, medium, large and total follicle numbers were 0.76, 0.40, 0.69 and 0.79, respectively. Medium size follicles presented only a small translucent area on the surface of the ovary, leading to an underestimate of numbers when categorised by surface evaluation. Counts of follicles visible on the surface of the ovaries of Bos indicus cows and their classification into size classes based on estimated diameter, are closely correlated with numbers of follicles in those size classes found at dissection of the ovary for small (8 mm) and total follicles but not for medium sized (4-8 mm) follicles. (C) 1997 Elsevier Science B.V.

Dendritic cells rapidly undergo apoptosis in vitro following culture with activated CD4(+) V alpha 24 natural killer T cells expressing CD40L

Human V alpha 24 natural killer T (V alpha 24NKT) cells are activated by -glycosylceramide-pulsed dendritic cells (DCs) in a CDld-dependent and T-cell receptor-mediated manner. There are two major subpopulations of V alpha 24NKT cells, CD4(-) CD8(-) V alpha 24NKT and CD4(+) V alpha 24NKT cells. We have recently shown that activated CD4(-) CD8 V alpha 24NKT cells have cytotoxic activity against DCs, but knowledge of the molecules responsible for cytotoxicity of V alpha 24NKT cells is currently limited. We aimed to investigate whether CD4(+) V alpha 24NKT cells also have cytotoxic activity against DCs and to determine the mechanisms underlying any observed cytotoxic activity. We demonstrated that activated CD4(+) V alpha 24NKT cells [CD40 ligand (CD40L) -positive] have cytotoxic activity against DCs (strongly CD40-positive), but not against monocytes (weakly CD40-positive) or phytohaemagglutinin blast T cells (CD40-negative), and that apoptosis of DCs significantly contributes to the observed cytotoxicity. The apoptosis of DCs following culture with activated CD4(+) V alpha 24NKT cells, but not with resting CD4(+) V alpha 24NKT cells (CD40L-negative), was partially inhibited by anti-CD40L mAb, Direct ligation of CD40 on the DCs by the anti-CD40 antibody also induced apoptosis of DCs. Our results suggest that CD40-CD40L interaction plays an important role in the induction of apoptosis of DCs following culture with activated CD4+ Va24NKT cells. The apoptosis of DCs from normal donors. triggered by the CD40-CD40L interaction, may contribute to the homeostatic regulation of the normal human immune system, preventing the interminable activation of activated CD4(+) V alpha 24NKT cells by virtue of apoptosis of DCs.

TRAIL expression by activated human CD4(+)V alpha 24NKT cells induces in vitro and in vivo apoptosis of human acute myeloid leukema cells

Human V alpha 24NKT cells are activated by alpha -galactosylceramide (alpha -GalCer)-pulsed dendritic cells in a CD1d-dependent and a T-cell receptor-mediated manner. Here, we demonstrate that CD4(+)V alpha 24NKT cells derived from a patient with acute myeloid leukemia (AML) M4 are phenotypically similar to those of healthy donors and, in common with those derived from healthy donors, express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) when the cells are activated by alpha -GalCer-pulsed dendritic cells but not prior to activation. We also show that myeloid that human activated CD4(+)V alpha 24NKT cells induced apoptosis of human leukemia cells in vivo. This is the first evidence that activated V alpha 24NKT cells express TRAIL and that TRAIL causes apoptosis of monocytic leukemia cells from patients with AML M4 in vitro and in vivo. Adoptive immune therapy with activated V alpha 24NKT cells, or other strategies to increase activated V alpha 24NKT cells in vivo, may be of benefit to patients with AML M4.