Andrea De Lerma Barbaro

Inflammation, inflammatory cells and angiogenesis: decisions and indecisions

Endothelial-immune cell cross-talk goes well beyond leukocyte and lymphocyte trafficking, since immune cells are able to intimately regulate vessel formation and function. Here we review the evidence that most immune cells are capable of polarization towards a dichotomous activity either inducing or inhibiting angiogenesis. In addition to the well-known roles of tumor associated macrophages, we find that neutrophils, myeloid-derived suppressor and dendritic cells clearly have the potential for influencing tumor angiogenesis. Further, the physiological functions of NK cells suggest that these cells may also show a potentially important role in pro- or anti-angiogenesis regulation within the tumor microenvironment. At the same time many angiogenic factors influence the activity and function of immune cells that generally favor tumor survival and tolerance. Thus the immune system itself represents a major pharmaceutical target and links angiogenesis inhibition to immunotherapy.

CIITA-Induced MHC Class II Expression in Mammary Adenocarcinoma Leads to a Th1 Polarization of the Tumor Microenvironment, Tumor Rejection, and Specific Antitumor Memory

Purpose: We have shown previously that the MHC class II–negative murine TS/A adenocarcinoma is rejected in vivo if induced to express MHC class II molecules by transfection of the MHC class II transactivator CIITA. In this study, we explored the immunologic basis of tumor rejection and the correlation between histopathology of tumor tissue and immune rejection. Experimental Design: Stable TS/A-CIITA transfectants were generated and injected into mice. In vivo cell depletion, immunohistochemistry of tumor tissues, and immune functional assays were done to assess the cellular and immunologic basis of rejection. Results: Ninety-two percent of mice injected with TS/A-CIITA rejected the tumor and were completely resistant to challenge with parental TS/A. Only CD4+ and CD8+ cells were required for rejection. The tumor microenvironment in TS/A-CIITA-injected mice changed dramatically when compared with the TS/A parental-injected mice. Rapid infiltration with CD4+ T cells followed by dendritic cells, CD8+ T cells, and granulocytes was observed. Importantly, TS/A-CIITA cells could act as antigen-presenting cells because they process and present nominal antigens to CD4+ T cells. Tumor-specific CD4+ T cells of TS/A-CIITA-injected mice had the functional characteristics of Th1 cells and produced IFN-γ and this was relevant for generation and maintenance of protective antitumor response, because IFN-γ knockout mice were no longer rejecting TS/A-CIITA tumor cells. Conclusion: CIITA-dependent MHC class II expression confers to TS/A tumor cells the capacity to act as a protective vaccine against the tumor by triggering tumor antigen presentation to T helper cells, antitumor polarization of cellular and soluble components of the tumor microenvironment, and establishment of antitumor immune memory.

Highly stable oligomerization forms of HIV‐1 Tat detected by monoclonal antibodies and requirement of monomeric forms for the transactivating function on the HIV‐1 LTR

The use of newly generated murine monoclonal antibodies directed against distinct epitopes of a functionally active, chemically synthesized HIV‐1 Tat protein has permitted the identification of several molecular forms including monomers, dimers and trimers. Dimers and trimers are particularly stable and resistant to strong reducing conditions. Through epitope mapping it has been possible to demonstrate that the major immunodominant epitope is contained within the basic region of the Tat protein and is lost after oligomerization of the molecule. In contrast, N‐terminal, C‐terminal and conformation‐dependent epitopes are still accessible to mAb specific recognition after Tat oligomerization. Moreover, by using a quantitative HIV‐LTR transactivation assay depending upon exogenous Tat, we could extrapolate the amount of functional Tat produced by cell lines stably transfected with the viral transactivator. More importantly, we could show that only the monomeric form of exogenous Tat is the relevant functional form acting in cells harbouring the HIV‐1 LTR promoter.

Therapy‐induced antitumor vaccination by targeting tumor necrosis factor‐α to tumor vessels in combination with melphalan

Treatment of tumor‐bearing mice with mouse (m)TNF‐α, targeted to tumor vasculature by the anti‐ED‐B fibronectin domain antibody L19(scFv) and combined with melphalan, induces a therapeutic immune response. Upon treatment, a highly efficient priming of CD4+ T cells and consequent activation and maturation of CD8+ CTL effectors is generated, as demonstrated by in vivo depletion and adoptive cell transfer experiments. Immunohistochemical analysis of the tumor tissue demonstrated massive infiltration of CD4+ and CD8+ T cells 6 days after treatment and much earlier in the anamnestic response to tumor challenge in cured mice. In fact, the curative treatment with L19mTNF‐α and melphalan resulted in long‐lasting antitumor immune memory, accompanied by a mixed Th1/Th2‐type response and significant in vitro tumor‐specific cytolytic activity. Finally, the combined treatment reduced the percentage and absolute number of CD4+CD25+ regulatory T cells in the tumor‐draining lymph nodes of mice responding to therapy, and this was associated with the establishment of protective immunity. These findings pave the way for alternative therapeutic strategies based on the targeted delivery of biological and pharmacological cytotoxic compounds that not only kill most of the tumor cells but, more importantly, trigger an effective and long‐lasting antitumor adaptive immune response.

The MHC class II transactivator: prey and hunter in infectious diseases

The MHC class II transcriptional activator (CIITA) is the major regulator of expression of MHC class II genes. Thus, CIITA plays a fundamental role in the regulation of the immune response. Here, we discuss our findings on the dual role of CIITA during infections, as the target (prey) for certain pathogens but the host effector (hunter) against other pathogens, including HIV-1. This dual role is placed in an evolutionary context as a rather peculiar example of a strategy used by pathogens to evade host defenses and a counteraction of the host to minimize the survival and spread of the pathogen.

The HLA class II transcriptional activator blocks the function of HIV-1 Tat and inhibits viral replication

The expression of HLA class II genes is under the control of a transcriptional activator, CIITA, encoded by the AIR‐1 locus. Here we show that CIITA inhibits HIV‐1 LTR transactivationmediated by Tat. The inhibition occurred when CIITA and Tat were transiently expressed in cells after transfection and, most importantly, when tat cDNA was transfected in cells expressing CIITA in a constitutive fashion and at physiological levels. Furthermore, CIITA inhibited the HIV‐1 LTR transactivation mediated by extracellular Tat protein. CIITA inhibition of Tat function could be reversed by overexpression of Cyclin T1, the cellular cofactor used by Tat to facilitate elongation of viral transcripts. CIITA inhibition of Tat function had a dramatic effect on HIV‐1 productive infection of human T cells because CIITA+ T cells supported very poorly, if any, viral replication. These results indicate that sustained expression of CIITA in HIV‐1‐susceptible targets may down‐regulate viral expression both in cells actively replicating the virus and in silently infected cells requiring exogenous Tat to reactivate virus from latency.

Distinct regulation of HLA class II and class I cell surface expression in the THP-1 macrophage cell line after bacterial phagocytosis

Expression of HLA and CD1b molecules was investigated in the THP‐1 macrophage cell line within 2 weeks following phagocytosis of mycobacteria or Escherichia coli. During the first 2 – 3 days, cell surface expression of HLA class II and CD1b was drastically down‐modulated, whereas HLA class I expression was up‐modulated. In the following days both HLA class II and CD1b expression first returned to normal, then increased and finally returned to normal with kinetics similar to that observed for the steadily increased HLA class I. The initial down‐modulation of HLA class II and CD1b cell surface antigens was absolutely dependent on phagocytosis of bacteria. Further studies indicated that initial HLA class II cell surface down‐modulation (1) was not due to reduced transcription or biosynthesis of mature HLA class II heterodimers, (2) was only partially, if at all, rescued by treatment with IFN‐γ, although both mRNA and corresponding intracellular proteins increased up to sixfold with respect to untreated cells, and (3) resulted in failure of THP‐1 cells to process and present mycobacterial antigens to HLA‐DR‐restricted antigen‐specific T cell lines. The existence of a transient block of transport of mature HLA class II heterodimers to the cell surface in the first days after phagocytosis of bacteria may have negative and positive consequences: it decreases APC function early but it may increase it later by favoring optimal loading of bacterial antigens in cellular compartments at high concentration of antigen‐presenting molecules.

HIV-1 Tat mutants in the cysteine-rich region downregulate HLA class II expression in T lymphocytic and macrophage cell lines.

Human macrophage and T cell lines were stably transfected with HIV‐1 wild‐type Tat or Tat mutants in the cysteine‐rich region displaying trans‐dominant negative effects on HIV‐1 life cycle. The expression of HLA class I and class II molecules was not affected by wild‐type Tat. Tat mutants, instead, profoundly down‐regulated in a dose‐dependent fashion the expression of class II, but not of class I, in both cell types by acting at the transcriptional level. Down‐regulation was manifested on constitutive and IFN‐γ‐induced class II gene expression and did not correlate with reduced transcription of the AIR‐1 gene product CIITA, the major transcriptional activator of class II genes, indicating that Tat mutants did not act by inhibiting AIR‐1 gene expression. Class II down‐modulation had important functional implications in macrophages, as both antigen processing and presenting capacity were inhibited. These results represent the first evidence that a modified HIV‐1 Tat product can act as a potent immunosuppressor by inhibiting the HLA class II expression necessary for triggering both cellular and humoral responses against pathogens. The use of these HIV‐1 Tat mutants also discloses new opportunities to investigate the molecular mechanisms underlying the coordinate HLA class II gene transcription.

The MHC class II transactivator (CIITA) mRNA stability is critical for the HLA class II gene expression in myelomonocytic cells

The human promyelocytic U937 cells express detectable levels of MHC class II (MHC‐II) molecules. Treatment with 12‐o‐‐tetradecanoyl phorbol 13‐acetate (TPA), inducing macrophage‐like differentiation, produces a dramatic decrease of MHC‐II expression as result of down‐modulation of the activation of immune response gene 1 (AIR‐1)‐encoded MHC‐II transactivator (CIITA). This event is specific, as MHC class I remains unaffected. Similar results are observed with U937 cells expressing an exogenous full‐length CIITA. Molecular studies demonstrate that TPA treatment affects the stability of CIITA mRNA rather than CIITA transcription. Importantly, cis‐acting elements within the distal 650 bp of the 1035‐bp 3′ untranslated region (3′UTR, nucleotides 3509–4543) are associated to transcript instability. Transcription inhibitors actinomycin D and 5,6‐dichlororibofuranosyl benzimidazole, and the translation inhibitor cycloheximide significantly rescue the accumulation of CIITA mRNA in TPA‐treated cells. A similar effect is also observed after treatment with staurosporine and the PKC‐specific inhibitor GF109203X. The instability of CIITA mRNA produced by TPA in U937 cells is not seen in B cells. These results demonstrate the presence of an additional level of control of MHC‐II expression in the macrophage cell lineage depending upon the control of CIITA mRNA stability, most likely mediated by differentiation‐induced, 3′UTR‐interacting factors which require kinase activity for their destabilizing function.

Irradiated CIITA-positive mammary adenocarcinoma cells act as a potent anti-tumor-preventive vaccine by inducing tumor-specific CD4+ T cell priming and CD8+ T cell effector functions.

In the present study, we investigated the possibility to use irradiated, non-replicating class II transcriptional activator (CIITA)-transfected tumor TS/A cells as a cell-based vaccine. Eighty-three percent of TS/A-CIITA-vaccinated mice were completely protected from tumor growth and the remaining 17% displayed significant reduction of tumor growth. In contrast, only 30% of mice injected with irradiated TS/A parental cells were protected from tumor growth, whereas the remaining 70% of animals remained unprotected. Immunity generated in the TS/A-CIITA-vaccinated mice correlated with an efficient priming of CD4(+) T cells and consequent triggering and maintenance of CD8(+) CTL effectors, as assessed by adoptive transfer assays. Important qualitative differences were observed between the two cell-based vaccines, as TS/A-CIITA-vaccinated mice developed a CTL response containing a large proportion of anti-gp70 AH1 epitope-specific cells, completely absent in TS/A-vaccinated mice, and a mixed T(h)1/T(h)2 type of response as opposed to a T(h)2 type of response in TS/A-vaccinated mice. Finally, in TS/A-CIITA-vaccinated mice, a statistically significant reduction in the percentage and absolute number of CD4(+) CD25(+) T regulatory cells as compared with those of untreated mice with growing tumors (P < 0.001) or mice vaccinated with TS/A parental cells were observed. These results let to envisage the use of CIITA-transfected non-replicating tumor cells as a vaccination strategy for prevention and, possibly, adjuvant immunotherapy in human settings.