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The article investigates the in vitro cell binding activity of such reagents and their antitumoral activity in immunocompetent murine model systems. It suggests that chimeric proteins combining a tumor antigen and specific recognition of antigen presenting cells (APC) in a single molecule are suitable for targeted delivery of antigens to professional APCs and might become valuable tools for cancer immunotherapy.

Purposeof Review: The main aim of this review is to summarize first-line therapy of nodal T-cell non-Hodgkin lymphoma., Recent Findings: Current treatment with CHOP chemotherapy results in poor outcomes in the majority of patients. However, there are advances within the field. First breakthrough is the ECHELON-2 trial which showed that the addition of brentuximab vedotin improves outcomes in anaplastic large cell lymphoma. However, other types of peripheral T-cell non-Hodgkin lymphoma were underrepresented with optimal treatment not known. Second breakthrough is an increase of autologous stem cell transplantation usage in the first complete metabolic remission, except in ALK + anaplastic large cell lymphoma, offering better disease control. Despite advances in the field, CHOP remains the standard treatment for the majority of these lymphomas, but multiple trials are underway with the aim to improve this unmet need in hematology and, hopefully, leading us to a new era in the treatment of peripheral T-cell lymphomas., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

ECHELON-1 study is a randomized open-labeled controlled trial investigating whether addition of brentuximab vedotin to chemotherapy offers benefit over the standard chemotherapy regimen in advanced Hodgkin lymphoma. After a median follow-up of 24.6 months, it has met its primary endpoint the reduction of modified progression-free survival being 23 percent. However, the beneficial effects have not been seen across all subgroups leading to further questions. The main aim of this review is to tackle these questions to provide the reader with in-depth insight of pros and cons of this novel, promising but ultimately controversial regimen., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

- The aim of this review is to present data on bendamustine, a non-cross resistant alkylating agent, alone or in combination for treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Bendamustine is currently approved for rituximab-resistant indolent NHL and CLL in patients not fit for conventional chemotherapy. Recent studies have shown superiority of bendamustine combination with rituximab (B-R) in first line treatment of indolent NHLs and mantle cell lymphoma, suggesting a shift of the standard of care in this setting. B-R regimen has also shown efficacy in relapsed setting suggesting the possible treatment option for patients failing conventional chemotherapy. In rituximab-resistant NHL, the recent GADOLIN study exploring the addition of obinutuzumab to bendamustine has yielded impressive result changing the standard of care in this hard-to-treat population. Concerning CLL, despite inferiority to the standard of care in young fit patients, as defined in CLL10 study, B-R has yielded a more beneficial toxicity profile and its use in first line treatment should be decided individually. In relapsed setting, the addition of ibrutinib to B-R has shown superior results compared to B-R alone, possibly changing the paradigm of treatment of relapsed CLL. In conclusion, bendamustine as a single agent or in combinations has shown activity with acceptable toxic profile in the treatment of patients with indolent NHLs or CLL without del(17p) mutation.

Even though Mycobacterium tuberculosis (Mtb) remains one of the top microbial killers, more than 90% of the 2 billion infected individuals never develop active tuberculosis (TB), indicating efficient immune control of infection in these individuals. Immune mechanisms promoting either control or reactivation of TB are incompletely understood. Kinetic analyses of T-cell responses against Mtb in C57BL/6 mice revealed surface expression of inducible co-stimulatory molecule (ICOS) on >30% of all CD4(+) T cells, suggesting a pivotal role of this costimulatory molecule of the CD28 family in TB control. Surprisingly, Mtb-infected ICOS(-/-) mice showed lower bacterial burden during the late chronic stage of infection as compared to WT controls. ICOS deficiency resulted in a reduced Mtb-specific CD8(+) T-cell response during late-stage infection. In contrast, the polyclonal CD4(+) Th1 response against Mtb was increased, most likely caused by diminished numbers and frequencies of Tregs. Thus, by altering effector T-cell populations differentially, ICOS signaling modulates TB control in the late stage of infection., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tuberculosis causes 2 million deaths per year, yet in most cases the immune response successfully contains the infection and prevents disease outbreak. Induced lymphoid structures associated with pulmonary granuloma are observed during tuberculosis in both humans and mice and could orchestrate host defense. To investigate whether granuloma perform lymphoid functions, mice lacking secondary lymphoid organs (SLO) were infected with Mycobacterium tuberculosis (MTB). As in WT mice, granuloma developed, exponential growth of MTB was controlled, and antigen-specific T-cell responses including memory T cells were generated in the absence of SLO. Moreover, adoptively transferred T cells were primed locally in lungs in a granuloma-dependent manner. T-cell activation was delayed in the absence of SLO, but resulted in a normal development program including protective subsets and functional recall responses that protected mice against secondary MTB infection. Our data demonstrate that protective immune responses can be generated independently of SLO during MTB infection and implicate local pulmonary T-cell priming as a mechanism contributing to host defense.

Tuberculosis (TB) remains a global health threat. Although it is generally accepted that TB results from intensive cross-talk between the host and the pathogen Mycobacterium tuberculosis, underlying mechanisms remain elusive. The first evidence of human polymorphisms related to susceptibilities to distinct M. tuberculosis lineages has been gathered. Confrontation of limited host resistance with heightened bacterial virulence forms a most hazardous combination. We investigated extreme combinations, confronting inducible nitric oxide synthase-deficient (iNOS(-/-)) and wild-type (WT) mice with 2 related M. tuberculosis strains that differ markedly in virulence, namely, the M. tuberculosis laboratory strains H37Rv and H37Ra. We provide evidence that deregulated chemokine signaling and excessive neutrophil necrosis contribute to disproportionate neutrophil influx and exacerbated TB in iNOS(-/-) mice infected with virulent M. tuberculosis (strain H37Rv), whereas resistant and susceptible mice controlled attenuated H37Ra equally well. Thus, a combination of host susceptibility and M. tuberculosis virulence determines the role of iNOS in the protection and control of inflammation.

Community-acquired pneumonia (CAP) is associated with high morbidity and mortality, and Streptococcus pneumoniae is the most prevalent causal pathogen identified in CAP. Impaired pulmonary host defense increases susceptibility to pneumococcal pneumonia. S. pneumoniae may up-regulate Toll-like receptor (TLR)-2 expression and activate TLR-2, contributing to pneumococcus-induced immune responses. In the current study, the course of severe murine pneumococcal pneumonia after pulmonary TLR-2-mediated immunostimulation with synthetic macrophage-activating lipopeptide-2 (MALP-2) was examined. Intratracheal MALP-2 application evoked enhanced proinflammatory cytokine and chemokine release, resulting in recruitment of polymorphonuclear neutrophils (PMN), macrophages, and lymphocytes into the alveolar space in WT, but not in TLR-2-deficient mice. In murine lungs as well as in human alveolar epithelial cells (A549), MALP-2 increased TLR-2 expression at both mRNA and protein level. Blood leukocyte numbers and populations remained unchanged. MALP-2 application 24 hours before intranasal pneumococcal infection resulted in increased levels of CCL5 associated with augmented leukocyte recruitment, and decreased levels of anti-inflammatory IL-10 in bronchoalveolar lavage fluid. Clinically, MALP-2-treated as compared with untreated mice showed increased survival, reduced hypothermia, and increased body weight. MALP-2 also reduced bacteremia and improved bacterial clearance in lung parenchyma, as examined by immunohistochemistry. In conclusion, pulmonary immunostimulation with MALP-2 before infection with S. pneumoniae improved local host defense and increased survival in murine pneumococcal pneumonia.

Activation of naive T cells is tightly controlled and depends on cognate interactions with professional antigen-presenting cells. We analyzed dependency on secondary lymphoid tissues for the activation of naive and memory CD4(+) and CD8(+) T cells following primary and secondary Listeria monocytogenes infection, respectively. In splenectomized lymphotoxin-beta receptor-deficient mice, lacking all secondary lymphoid tissues, oral infection with L. monocytogenes failed to induce bacteria-specific CD4(+) and CD8(+) T cell responses. Treatment of splenectomized wild-type mice with FTY720, a drug that prevents egress of T cells from lymph nodes, also reduced T cell responses after oral L. monocytogenes infection and blocked T cell responses after intravenous infection. FTY720-treated wild-type and lymphotoxin-beta receptor-deficient mice show only slightly impaired recall responses. However, T cell responses were profoundly inhibited when mice were splenectomized subsequently to recovery from primary infection. T cell transfer experiments demonstrated that the impaired secondary T cell response was not simply due to removal of a large fraction of memory T cells by splenectomy. Overall, these results indicate that not only primary T cell responses, but also secondary T cell responses, highly depend on the lymphoid environment for effective activation.

Background: The prevalence of asthma and allergic diseases in children has been increasing worldwide over the past decades. The ISAAC Phase I results supplies valuable information on the worldwide variations in the prevalence of these diseases. Although ISAAC Phase I was completed in 56 countries, not all regions of Croatia were covered. Because of Croatia's high regional diversity, the aim was to explore the prevalence of asthma, allergic rhinitis/rhinoconjuctivitis, and atopic eczema symptoms in the Medimurje region in northern Croatia and compare the results with data from other regions in Croatia and other countries., Material/methods: The study was undertaken between January and April 2005 among 12- to 14-year-old children in 27 elementary schools. Data were collected using the standardized ISAAC written and asthma video questionnaires., Results: A total of 3111 children participated in the study, with a participation rate of 94.33%. 27.6% of the children had symptoms of allergic diseases at some time in their life. Estimated lifetime (ever) prevalence of symptoms were: wheezing 11.86%, allergic rhinitis symptoms 12.21%, and atopic dermatitis symptoms 7.01%. Estimated 12-month prevalence rates were: wheezing 5.11%, allergic rhinitis symptoms 10.87%, allergic rhinoconjunctivitis 7.14%, and atopic dermatitis symptoms 5.34%., Conclusions: Compared with previous studies conducted in other Croatian regions (the city of Zagreb and a northern Adriatic region) using similar methods, the prevalence of asthma and allergic rhinitis was lower than in the northern Adriatic region but comparable with that in the city of Zagreb, and of atopic dermatitis symptoms in the same range.

Mycobacterium tuberculosis remains one of the top microbial killers of humans causing approximately 2 million deaths annually. More than 90% of the 2 billion individuals infected never develop active disease, indicating that the immune system is able to generate mechanisms that control infection. However, the immune response generally fails to achieve sterile clearance of bacilli. Using adoptive cell transfer into C57BL/6J-Rag1(tm1Mom) mice (Rag1(-/-)), we show that regulatory T cells prevent eradication of tubercle bacilli by suppressing an otherwise efficient CD4+ T cell response. This protective CD4+ T cell response was not correlated with increased numbers of IFN-gamma- or TNF-alpha-expressing cells or general expression levels of IFN-gamma or inducible NO synthase in infected organs compared with wild-type C57BL/6 animals. Furthermore, suppression of protection by cotransferred regulatory T cells was neither accompanied by a general increase of IL-10 expression nor by higher numbers of IL-10-producing CD4+ T cells.

The adaptor protein linker for activation of T cells (LAT) is an important transducer of extracellular T cell stimuli. In mice with a point mutation in LAT (LatY136F), TCR signaling is substantially compromised and LatY136F T cells are unresponsive to CD3 cross-linking in vitro. Nevertheless, LatY136F mice develop a polyclonal lymphoproliferation of CD4(+) T cells, which display a T(h)2-polarized effector phenotype. In this study, LatY136F mice were infected with the intracellular bacterium Listeria monocytogenes and the antigen-specific responses of T cells were determined. Both CD4(+) and CD8(+) LatY136F T cells were unresponsive to L. monocytogenes infection. In contrast, when CD4(+) T cells from wild-type mice were adoptively transferred into LatY136F hosts, they responded normally to L. monocytogenes, indicating that the LatY136F milieu permits T(h)1 responses. Furthermore, we analyzed whether the infection would influence the capacity of LatY136F CD4(+) T cells to produce IL-4 and IFN-gamma. While L. monocytogenes infection results in T(h)1-type T cell responses in wild-type animals, we found that it did not shift the strong T(h)2 polarization of LatY136F T cells towards a T(h)1 pattern. In conclusion, our results suggest that the activation and T(h)2 polarization of the LatY136F CD4(+) T cells is not influenced by infection with an intracellular pathogen known to induce robust T(h)1 responses, and is thus likely driven by T cell intrinsic mechanisms.

Effective priming of T cell responses depends on cognate interactions between naive T cells and professional antigen-presenting cells (APCs). This contact is the result of highly coordinated migration processes, in which the chemokine receptor CCR7 and its ligands, CCL19 and CCL21, play a central role. We used the murine Listeria monocytogenes infection model to characterize the role of the CCR7/CCR7 ligand system in the generation of T cell responses during bacterial infection. We demonstrate that efficient priming of naive major histocompatibility complex (MHC) class Ia-restricted CD8+ T cells requires CCR7. In contrast, MHC class Ib-restricted CD8+ T cells and MHC class II-restricted CD4+ T cells seem to be less dependent on CCR7; memory T cell responses are independent of CCR7. Infection experiments with bone marrow chimeras or mice reconstituted with purified T cell populations indicate that CCR7 has to be expressed on CD8+ T cells and professional APCs to promote efficient MHC class Ia-restricted T cell priming. Thus, different T cell subtypes and maturation stages have discrete requirements for CCR7.

Activation of T cells by professional APCs that present peptide epitopes of tumor-associated Ags is critical for the induction of cell-mediated immunity against tumors. To facilitate targeted delivery of the ErbB2 (HER2, neu) tumor Ag to APCs in vivo, we have generated chimeric proteins that contain the extracellular domain of CTLA-4 for binding to B7 molecules on the APC surface, which is genetically fused to a human ErbB2 fragment as an antigenic determinant. Bacterially expressed CTLA-4-ErbB2 fusion protein and a similar molecule harboring in addition the translocation domain of Pseudomonas exotoxin A as an endosome escape function displayed specific binding to B7-expressing cells, followed by protein internalization and intracellular degradation. Vaccination of BALB/c mice with the fusion proteins resulted in the induction of ErbB2-specific CD8(+) T cells and CTL-dependent protection from subsequent challenge with ErbB2-expressing but not ErbB2-negative murine renal carcinoma cells. In a therapeutic setting, injection of CTLA-4-ErbB2 protein vaccines caused rejection of established ErbB2-expressing tumors. Thereby, immunological memory was induced, leading to long-term systemic immunity and protection against rechallenge several months later. Our results demonstrate that these chimeric protein vaccines are effective tools for the induction of ErbB2-specific, T cell-mediated immunity.

We addressed the question of whether protective immunity induced by natural infection with Mycobacterium tuberculosis and that induced by vaccination with Mycobacterium bovis bacille Calmette-Guerin (BCG) differ in the murine model. We infected mice with M. tuberculosis Erdman, cured them by chemotherapy, and subsequently reinfected them with a low dose of M. tuberculosis H37Rv. The course of tuberculosis was compared with that in mice previously vaccinated with BCG Danish 1331. Protection against postprimary M. tuberculosis infection did not differ significantly between the 2 groups. After challenge infection, numbers of interferon- gamma -positive splenocytes did not differ between mice with primary infection and vaccinated mice. Splenocytes from primary M. tuberculosis-infected mice conferred marginally higher protection than did those from BCG-vaccinated mice. Serum transfer did not protect against reinfection in either group. Our data emphasize that natural infection with M. tuberculosis and vaccination with BCG do not differ in their capacity to induce protective immunity against tuberculosis and support the notions that reinfection contributes to the development of active disease and that any novel vaccine against tuberculosis has to perform better than both vaccination with BCG and immunity evoked by natural infection.

A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a phosphatidylinositol mannoside (PIM) fulfilled the requirements for CD1d binding and NKT cell stimulation. This PIM activated human and murine NKT cells via CD1d, thereby triggering antigen-specific IFN-gamma production and cell-mediated cytotoxicity, and PIM-loaded CD1d tetramers identified a subpopulation of murine and human NKT cells. This phospholipid, therefore, represents a mycobacterial antigen recognized by T cells in the context of CD1d.

Immunization of mice with nonviable Listeria monocytogenes generates an insufficient CD8(+) T cell response and consequently only limited protection against subsequent L. monocytogenes infection. We have recently demonstrated that depletion of regulatory CD4(+) T cells during immunization significantly enhances CD8(+) T cell responses. In the present study, we determined the impact of CD4(+) T cell depletion on the CD8(+) T cell response against heat-killed Listeria. Treatment of mice with anti-CD4 mAb during boost immunization with heat-killed Listeria significantly increased numbers of Listeria-specific CD8(+) T cells and improved protection against subsequent infection with L. monocytogenes. During challenge infection, numbers of Listeria-specific CD8(+) T cells were enhanced, and these cells expressed effector functions in terms of IFN-gamma production. In summary, we demonstrate that combining nonviable L. monocytogenes vaccination and CD4(+) T cell depletion improves generation of long-lasting and functional Listeria-specific CD8(+) memory T cells.

Infection of mice with Listeria monocytogenes induces a strong CD8+ T cell response, which is critical for the control of bacteria and for protection against re-infection. We analyzed the CD8+ T cell response in different intestinal tissues following oral and intravenous (i.v.) L. monocytogenes infection. After oral infection, bacterial titers in small intestine and large intestine, and the listeria-specific CD8+ T cell response in the mucosa of both parts of the intestine, were highly correlated. Oral infection of CD28-deficient mice revealed that this response was strictly dependent on CD28 costimulation. Significant listeria-specific CD8+ T cell responses also occurred in all intestinal tissues analyzed after i.v. infection or after DNA vaccination, indicating that the accumulation of listeria-specific CD8+ T cells in these tissues only partially depends on local antigen presentation and inflammation.

CD8(+) T-cells are a major source for the production of non-cytolytic factors that inhibit HIV-1 replication. In order to characterize further these factors, we analyzed gene expression profiles of activated CD8(+) T-cells using a human cDNA expression array containing 588 human cDNAs. mRNA for the chemokine I-309 (CCL1), the cytokines granulocyte-macrophage colony-stimulating factor and interleukin-13, and natural killer cell enhancing factors (NKEF) -A and -B were up-regulated in bulk CD8(+) T-cells from HIV-1 seropositive individuals compared with seronegative individuals. Recombinant NKEF-A and NKEF-B inhibited HIV-1 replication when exogenously added to acutely infected T-cells at an ID(50) (dose inhibiting HIV-1 replication by 50%) of approximately 130 nm (3 microg/ml). Additionally, inhibition against dual-tropic simian immunodeficiency virus and dual-tropic simian-human immunodeficiency virus was found. T-cells transfected with NKEF-A or NKEF-B cDNA were able to inhibit 80-98% HIV-1 replication in vitro. Elevated plasma levels of both NKEF-A and NKEF-B proteins were detected in 23% of HIV-infected non-treated individuals but not in persons treated with highly active antiviral therapy or uninfected persons. These results indicate that the peroxiredoxin family members NKEF-A and NKEF-B are up-regulated in activated CD8(+) T-cells in HIV infection, and suggest that these antioxidant proteins contribute to the antiviral activity of CD8(+) T-cells.

CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.

The inducible costimulator protein (ICOS) was recently identified as a costimulatory molecule for T cells. Here we analyze the role of ICOS for the acquired immune response of mice against the intracellular bacterium Listeria monocytogenes. During oral L. monocytogenes infection, low levels of ICOS expression were detected by extracellular and intracellular Ab staining of Listeria-specific CD4(+) and CD8(+) T cells. Blocking of ICOS signaling with a soluble ICOS-Ig fusion protein markedly impaired the Listeria-specific T cell responses. Compared with control mice, the ICOS-Ig treated mice generated significantly reduced numbers of Listeria-specific CD8(+) T cells in spleen and liver, as determined by tetramer and intracellular cytokine staining. In contrast, the specific CD8(+) T cell response in the intestinal mucosa did not appear to be impaired by the ICOS-Ig treatment. Analysis of the CD4(+) T cell response revealed that ICOS-Ig treatment also affected the specific CD4(+) T cell response. When restimulated with listerial Ag in vitro, reduced numbers of CD4(+) T cells from infected and ICOS-Ig-treated mice responded with IFN-gamma production. The impaired acquired immune response in ICOS-Ig treated mice was accompanied by their increased susceptibility to L. monocytogenes infection. ICOS-Ig treatment drastically enhanced bacterial titers, and a large fraction of mice succumbed to the otherwise sublethal dose of infection. Thus, ICOS costimulation is crucial for protective immunity against the intracellular bacterium L. monocytogenes.

The immune response against the intracellular bacterium Listeria monocytogenes involves both CD4(+) and CD8(+) T cells. We used the MHC class II-presented peptide listeriolysin(189-201) to characterize the organ-specific CD4(+) T cell response during infection. Systemic listeriosis resulted in a strong peptide-specific CD4(+) T cell response with frequencies of 1/100 and 1/30 CD4(+) splenocytes at the peak of primary and secondary response, respectively. This response was not restricted to lymphoid organs, because we detected specific CD4(+) T cells in all tissues analyzed. However, the tissue distribution of the T cell response was dependent on the route of infection. After i.v. infection, the strongest CD4(+) T cell response and the highest levels of memory cells were observed in spleen and liver, the major sites of L. monocytogenes replication. After oral infection, we detected a strong response in the liver, the lamina propria, and the intestinal epithelium. These tissues also harbored the highest frequencies of listeriolysin(189-201)-specific CD4(+) memory T cells 5-8 wk post oral infection. Our results show that kinetics and magnitude of the CD4(+) T cell response and the accumulation of CD4(+) memory T cells depend on the route of infection and are regulated in a tissue-specific way.

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers.

Live recombinant vaccines expressing defined pathogen-derived Ags represent powerful candidates for future vaccination strategies. In this study, we report on the differential induction of protective cell-mediated immunity elicited by different recombinant Mycobacterium bovis Bacille Calmette-Guérin (BCG) strains displaying p60 Ag of Listeria monocytogenes in secreted, cytosolic, or membrane-attached form for T cell recognition. Anti-listerial protection evoked by the membrane-linked p60 lipoprotein of rBCG Mp60 and that of the p60 derivative secreted by rBCG Sp60-40 were nearly equal, whereas cytosolic p60 displayed by rBCG Np60 failed to protect mice from listeriosis. In vivo depletion of CD4 or CD8 T cell subpopulations in rBCG Mp60-vaccinated mice before listerial challenge revealed interactions of both T cell subsets in anti-listerial protection. In rBCG Sp60-40-vaccinated animals, CD4 T cells predominantly contributed to anti-listerial control as shown by the failure of anti-CD8 mAb treatment to impair the outcome of listeriosis in rBCG Sp60-40-vaccinated mice after L. monocytogenes challenge. Hence, differential Ag display by rBCG influences cell-mediated immunity, which in turn may impact vaccine efficacy due to the different requirements of CD4 or CD8 T cells for pathogen elimination.

Infection of mice with the intracellular bacterium Listeria monocytogenes results in a strong CD8(+) T cell response that is critical for efficient control of infection. We used CD28-deficient mice to characterize the function of CD28 during Listeria infection, with a main emphasis on Listeria-specific CD8(+) T cells. Frequencies and effector functions of these T cells were determined using MHC class I tetramers, single cell IFN-gamma production and Listeria-specific cytotoxicity. During primary Listeria infection of CD28(-/-) mice we observed significantly reduced numbers of Listeria-specific CD8(+) T cells and only marginal levels of specific IFN-gamma production and cytotoxicity. Although frequencies were also reduced in CD28(-/-) mice during secondary response, we detected a considerable population of Listeria-specific CD8(+) T cells in these mice. In parallel, IFN-gamma production and cytotoxicity were observed, revealing that Listeria-specific CD8(+) T cells in CD28(-/-) mice expressed normal effector functions. Consistent with their impaired CD8(+) T cell activation, CD28(-/-) mice suffered from exacerbated listeriosis both after primary and secondary infection. These results demonstrate participation of CD28 signaling in the generation and expansion of Ag-specific CD8(+) T cells in listeriosis. However, Ag-specific CD8(+) T cells generated in the absence of CD28 differentiated into normal effector and memory T cells.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) mediate immunologic selection pressure by both cytolytic and noncytolytic mechanisms. Non cytolytic mechanisms include the release of beta-chemokines blocking entry of R5 HIV-1 strains. In addition, CD8(+) cells inhibit X4 virus isolates via release of as yet poorly characterized soluble factors. To further characterize these factors, we performed detailed analysis of CTL as well as bulk CD8(+) T lymphocytes from six HIV-1-infected individuals and from six HIV-1-seronegative individuals. Kinetic studies revealed that secreted suppressive activities of HIV-1-specific CTL and bulk CD8(+) T lymphocytes from all HIV-1-infected persons are significantly higher than that of supernatants from seronegative controls. The suppressive activity could be blocked by monensin and brefeldin A, was heat labile, and appeared in a pattern different from that of secretion of chemokines (MDC, I-309, MIP-1alpha, MIP-1beta, and RANTES), cytokines (gamma interferon, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor), and interleukins (interleukin-13 and interleukin-16). This suppression activity was characterized by molecular size exclusion centrifugation and involves a suppressive activity of >50 kDa which could be bound to heparin and a nonbinding inhibitory activity of <50 kDa. Our data provide a functional link between CD8(+) cells and CTL in the noncytolytic inhibition of HIV-1 and suggest that suppression of X4 virus is mediated through proteins. The sizes of the proteins, their affinity for heparin, and the pattern of release indicate that these molecules are not chemokines.