Dalia Khalil

CD40 and CD86 upregulation with divergent CMRF44 expression on blood dendritic cells in inflammatory bowel diseases.

OBJECTIVE:Dendritic cells (DC) are the only antigen-presenting cells that can activate naïve T lymphocytes and initiate a primary immune response. They are also thought to have a role in immune tolerance. DC traffic from the blood to peripheral tissue where they become activated. They then present antigen and the costimulating signals necessary to initiate an immune response. In this study, we investigated the number, subsets, and activation pattern of circulating and intestinal DC from patients with clinically mild ulcerative colitis (UC) or Crohns disease.METHODS:Patients were recruited, if they were not taking immunosuppressive therapy, and were assessed for clinical severity of their disease using for UC, the Clinical Activity Index, and for Crohns disease, the Crohns Disease Activity Index. Blood CD11c+ and CD11c− DC subsets, expression of costimulatory antigens, CD86 and CD40, and the early differentiation/activation antigen, CMRF44, were enumerated by multicolor flow cytometry of lineage negative (lin−= CD3−, CD19−, CD14−, CD16−) HLA-DR+ DC. These data were compared with age-matched healthy and the disease control groups of chronic noninflammatory GI diseases (cGI), acute noninflammatory GI diseases (aGI), and chronic non-GI inflammation (non-GI). In addition, cryostat sections of colonoscopic biopsies from healthy control patients and inflamed versus noninflamed gut mucosa of inflammatory bowel disease (IBD) patients were examined for CD86+ and CD40+lin− cells.RESULTS:Twenty-one Crohns disease and 25 UC patients, with mean Crohns Disease Activity Index of 98 and Clinical Activity Index of 3.1, and 56 healthy controls, five cGI, five aGI, and six non-GI were studied. CD11c+ and CD11c− DC subsets did not differ significantly between Crohns, UC, and healthy control groups. Expression of CD86 and CD40 on freshly isolated blood DC from Crohns patients appeared higher (16.6%, 31%) and was significantly higher in UC (26.6%, 46.3%) versus healthy controls (5.5%, 25%) (p = 0.004, p = 0.012) and non-GI controls (10.2%, 22.8%) (p = 0.012, p = 0.008), but not versus cGI or aGI controls. CD86+ and CD40+ DC were also present in inflamed colonic and ileal mucosa from UC and Crohns patients but not in noninflamed IBD mucosa or normal mucosa. Expression of the CMRF44 antigen was low on freshly isolated DC, but it was upregulated after 24-h culture on DC from all groups, although significantly less so on DC from UC versus Crohns or healthy controls (p = 0.024). The CMRF44+ antigen was mainly associated with CD11c+ DC, and in UC was inversely related to the Clinical Activity Index (r = −0.69, p = 0.0002).CONCLUSIONS:There is upregulation of costimulatory molecules on blood DC even in very mild IBD but surprisingly, there is divergent expression of the differentiation/activation CMRF44 antigen. Upregulation of costimulatory molecules and divergent expression of CMRF44 in blood DC was also apparent in cGI and aGI but not in non-GI or healthy controls, whereas intestinal CD86+ and CD40+ DC were found only in inflamed mucosa from IBD patients. Persistent or distorted activation of blood DC or divergent regulation of costimulatory and activation antigens may have important implications for gut mucosal immunity and inflammation.

FLT3-Ligand Treatment of Humanized Mice Results in the Generation of Large Numbers of CD141+ and CD1c+ Dendritic Cells In Vivo

We established a humanized mouse model incorporating FLT3-ligand (FLT3-L) administration after hematopoietic cell reconstitution to investigate expansion, phenotype, and function of human dendritic cells (DC). FLT3-L increased numbers of human CD141+ DC, CD1c+ DC, and, to a lesser extent, plasmacytoid DC (pDC) in the blood, spleen, and bone marrow of humanized mice. CD1c+ DC and CD141+ DC subsets were expanded to a similar degree in blood and spleen, with a bias toward expansion of the CD1c+ DC subset in the bone marrow. Importantly, the human DC subsets generated after FLT3-L treatment of humanized mice are phenotypically and functionally similar to their human blood counterparts. CD141+ DC in humanized mice express C-type lectin-like receptor 9A, XCR1, CADM1, and TLR3 but lack TLR4 and TLR9. They are major producers of IFN-λ in response to polyinosinic-polycytidylic acid but are similar to CD1c+ DC in their capacity to produce IL-12p70. Although all DC subsets in humanized mice are efficient at presenting peptide to CD8+ T cells, CD141+ DC are superior in their capacity to cross-present protein Ag to CD8+ T cells following activation with polyinosinic-polycytidylic acid. CD141+ DC can be targeted in vivo following injection of Abs against human DEC-205 or C-type lectin-like receptor 9A. This model provides a feasible and practical approach to dissect the function of human CD141+ and CD1c+ DC and evaluate adjuvants and DC-targeting strategies in vivo.

Blood monocytes, myeloid dendritic cells and the cytokines interleukin (IL)-7 and IL-15 maintain human CD4+ T memory cells with mixed helper/regulatory function

The number and function of human T cells in the periphery are regulated by homeostatic signals received from antigen‐presenting cells (APCs) and the common gamma chain (γc) cytokines interleukin (IL)‐7 and IL‐15. We found that, in the absence of introduced antigen, blood monocytes or myeloid dendritic cells (MDCs) in the presence of IL‐7 and IL‐15 (IL‐7/IL‐15) can regulate CD4+ T memory (Tm) cell numbers by polyclonal cell proliferation. The dynamics of CD4+ Tm cell proliferation, in the presence of IL‐7/IL‐15, was dependent on contact with MDCs and to a lesser extent on contact with monocytes. IL‐7/IL‐15 either alone or combined with monocytes or MDCs enhanced the proportion of CD4+ Tm cells with activated and effector phenotype and diminished the helper function of CD4+ Tm cells. These CD4+ Tm cells, preconditioned with IL‐7/IL‐15 alone or with monocytes or MDCs and IL‐7/IL‐15, reduced T cell‐dependent immunoglobulin M (IgM) and IgG responses. This appeared to be a contact‐dependent effect involving a reduction in antibody‐producing CD27+ B memory cells, but contact‐independent suppression by soluble factors also contributed to the antibody‐producing capacity of CD27+ B memory cells. These results indicate that blood monocytes, MDCs and the cytokines IL‐7/IL‐15 contribute to homeostasis of CD4+ Tm cells by regulating their number, activation state and helper/suppressor (regulatory) function. In healthy individuals, this mode of regulating CD4+ Tm cell homeostasis may provide a basis for the control of autoimmune responses.

Myeloma-Induced Alloreactive T Cells Arising in Myeloma-Infiltrated Bones Include Double-Positive CD8+CD4+ T Cells: Evidence from Myeloma-Bearing Mouse Model

The graft-versus-myeloma (GVM) effect represents a powerful form of immune attack exerted by alloreactive T cells against multiple myeloma cells, which leads to clinical responses in multiple myeloma transplant recipients. Whether myeloma cells are themselves able to induce alloreactive T cells capable of the GVM effect is not defined. Using adoptive transfer of T naive cells into myeloma-bearing mice (established by transplantation of human RPMI8226-TGL myeloma cells into CD122+ cell-depleted NOD/SCID hosts), we found that myeloma cells induced alloreactive T cells that suppressed myeloma growth and prolonged survival of T cell recipients. Myeloma-induced alloreactive T cells arising in the myeloma-infiltrated bones exerted cytotoxic activity against resident myeloma cells, but limited activity against control myeloma cells obtained from myeloma-bearing mice that did not receive T naive cells. These myeloma-induced alloreactive T cells were derived through multiple CD8+ T cell divisions and enriched in double-positive (DP) T cells coexpressing the CD8αα and CD4 coreceptors. MHC class I expression on myeloma cells and contact with T cells were required for CD8+ T cell divisions and DP-T cell development. DP-T cells present in myeloma-infiltrated bones contained a higher proportion of cells expressing cytotoxic mediators IFN-γ and/or perforin compared with single-positive CD8+ T cells, acquired the capacity to degranulate as measured by CD107 expression, and contributed to an elevated perforin level seen in the myeloma-infiltrated bones. These observations suggest that myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones are enriched with DP-T cells equipped with cytotoxic effector functions that are likely to be involved in the GVM effect.

Compartmentalization of allogeneic T-cell responses in the bone marrow and spleen of humanized NOD/SCID mice containing activated human resident myeloid dendritic cells.

OBJECTIVE Human allogeneic (allo)-T-cell responses within recipient lymphoid tissues and the degree to which they are altered in the presence of activated tissue-resident dendritic cells (DC) remain unknown. This study examined allo-T-cell recruitment and the early allo-T-cell responses that occur in the bone marrow (BM) and spleen (SP) of humanized (hu) nonobese diabetic (NOD)/severe combined immunodeficient (SCID) recipients containing activated human tissue-resident myeloid DC (MDC). MATERIALS AND METHODS Human naïve allo-T cells were transferred into polyinosinic:polycytidylic acid [poly(I:C)]-treated or untreated huNOD/SCID recipients containing human tissue-resident DC derived from transplanted CD34(+) cells. Activation of human tissue-resident MDC mediated by poly(I:C) treatment, recruitment, proliferation, and effector differentiation of allo-T cells in the BM and SP of huNOD/SCID recipients were analyzed in vivo by flow cytometry. RESULTS Poly(I:C) treatment induced transient activation of human MDC within a maximum of 8 hours, as evidenced in the BM by an increased proportion of MDC-expressing CD86 while in the SP by MDC expressing CD86 and producing interleukin-12. Poly(I:C)-pretreated huNOD/SCID recipients showed changes in the recruitment of allo-T cells in the BM and SP and developed different allo-T cell responses within the BM and SP compartments. In the BM, allo-T cells underwent multiple divisions and increased numbers of interferon-gamma(+) and tumor necrosis factor-alpha(+) effector cells, while the majority of splenic allo-T cells underwent a single division and had fewer effector allo-T cells. CONCLUSIONS Our experimental transplantation model demonstrates that early allo-T-cell responses are regulated by compartmentalization in the BM and secondary lymphoid tissues; events potentially occurring after allotransplantation in human recipients.

Type 1 Diabetic Children and Siblings Share a Decrease in Dendritic Cell and Monocyte Numbers but are Differentiated by Expansion of CD4 + T Cells Expressing IL-17

Type 1 diabetes (T1D) is an autoimmune disease characterised by multiple defects of immune cells which allow the expansion of pathogenic β cell-specific T effector cells and subsequent T1D development. We performed immunomonitoring assays on blood immune cells in T1D children, their siblings and controls with the supposition that the results would elucidate the sequence of abnormalities in peripheral immune cells leading to the progression of autoimmunity from non-diabetic siblings to diabetic children. We assessed myeloid dendritic cell (MDC), plasmacytoid (P)DC, monocyte, CD4+T and CD8+T cell compartments, and cytokine expression in CD4+T cells and CD8+T cells, by polychromatic flow cytometry in erythrocyte-lysed fresh blood. The numbers of CD16+MDC, PDC and CD16+ monocytes were similarly decreased in diabetic children and their siblings. Whereas the numbers of CD16-CD141-MDC, CD16-CD141+MDC, CD16- monocytes, T naive (CD45RO-CD62L+), T central memory (CD45RO+CD62L+), T effector memory (CD45RO+CD62L-), T terminally-differentiated effector (CD45ROCD62L-) and T regulatory cells (CD4+CD25+127lo/- or CD4+Foxp3+ cells) were not affected in diabetic children or their siblings. Furthermore, analysis of cytokine expression in CD4+T cells and CD8+T cell revealed an increased proportion of CD4+T cells expressing IL-17, differentiating diabetic children from their non-diabetic siblings. Our data suggest that diabetic children and their siblings suffer a considerable reduction of blood immune cells involving CD16+MDC, PDC and CD16+ monocytes that may result from shared genetic or environmental factors. However, this reduction of blood immune cells requires combination with the proinflammatory cytokine IL-17 to allow disease expression in diabetic children.

The CMRF58 antibody recognizes a subset of CD123hi dendritic cells in allergen-challenged mucosa

CD123hi CD11c− dendritic cells (CD123hi DC) are a distinct subset of human DC present in bone marrow, blood, lymphoid organs, and peripheral tissues. Pathogen stimulation, cytokine, or CD40 ligation induces CD123hi DC maturation, involving a shift from their innate immune to cognate antigen‐presenting functions. In this study, we revealed that blood CD123hi DC in the presence of cytokine (granulocyte macrophage‐colony stimulating factor and interleukin‐3) undergo progressive, step‐wise maturation through an “early” stage, delineated by expression of the antigen detected by the new monoclonal antibody CMRF58 (CD123hiCMRF58+CD40−CD86−CD83−) to the “late” stage with costimulatory antigen expression (CD123hiCMRF58+CD40+CD86+CD83+/−). In this early stage, cytokine‐maintained CD123hi DC do not display changes in their morphology, no longer produce interferon‐α (IFN‐α) in response to bacteria, and develop the capacity to induce proliferation and polarization of allogeneic T cells. CD123hiCMRF58+ DC, phenotypically similar to in vitro cytokine‐maintained CD123hi DC, were not detected in tonsil but are present in allergen‐challenged nasal mucosa of allergic individuals. Thus, CD123hi DC in certain tissue environments such as allergen‐challenged nasal mucosa share a common CD123hiCMRF58+ phenotype with in vitro cytokine‐maintained blood CD123hi DC characterized by lack of IFN‐α production.