The Journal of Experimental Medicine

Wang, Ji Ming; Oppenheim, Joost J.

doi: 10.1084/jem.190.5.591pmid: 10477544

Alfano, Massimo; Schmidtmayerova, Helena; Amella, Carol-Ann; Pushkarsky, Tatiana; Bukrinsky, Michael

doi: 10.1084/jem.190.5.597pmid: 10477545

Infection of target cells by HIV-1 requires initial binding interactions between the viral envelope glycoprotein gp120, the cell surface protein CD4, and one of the members of the seven-transmembrane G protein–coupled chemokine receptor family. Most primary isolates (R5 strains) use chemokine receptor CCR5, but some primary syncytium-inducing, as well as T cell line–adapted, strains (X4 strains) use the CXCR4 receptor. Signaling from both CCR5 and CXCR4 is mediated by pertussis toxin (PTX)-sensitive G i proteins and is not required for HIV-1 entry. Here, we show that the PTX holotoxin as well as its binding subunit, B-oligomer, which lacks G i -inhibitory activity, blocked entry of R5 but not X4 strains into primary T lymphocytes. Interestingly, B-oligomer inhibited virus production by peripheral blood mononuclear cell cultures infected with either R5 or X4 strains, indicating that it can affect HIV-1 replication at both entry and post-entry levels. T cells treated with B-oligomer did not initiate signal transduction in response to macrophage inflammatory protein (MIP)-1β or RANTES (regulated upon activation, normal T cell expressed and secreted); however, cell surface expression of CCR5 and binding of MIP-1β or HIV-1 to such cells were not impaired. The inhibitory effect of B-oligomer on signaling from CCR5 and on entry of R5 HIV-1 strains was reversed by protein kinase C (PKC) inhibitors, indicating that B-oligomer activity is mediated by signaling events that involve PKC. B-oligomer also blocked cocapping of CCR5 and CD4 induced by R5 HIV-1 in primary T cells, but did not affect cocapping of CXCR4 and CD4 after inoculation of the cultures with X4 HIV-1. These results suggest that the B-oligomer of PTX cross-deactivates CCR5 to impair its function as a coreceptor for HIV-1. CCR5 signal transduction G i protein receptor capping receptor desensitization Footnotes 1used in this paper: CCR, CC chemokine receptor; CXCR, CXC chemokine receptor; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; PKC, protein kinase C; PTX, pertussis toxin; RANTES, regulated upon activation, normal T cell expressed and secreted; SDF, stroma-derived factor Submitted: 22 January 1999 Revision requested 1 July 1999 Accepted: 6 July 1999

Iijima, Hideki; Takahashi, Ichiro; Kishi, Daisuke; Kim, Jin-Kyung; Kawano, Sunao; Hori, Masatsugu; Kiyono, Hiroshi

doi: 10.1084/jem.190.5.607pmid: 10477546

T cell receptor α chain–deficient (TCR-α −/− ) mice are known to spontaneously develop inflammatory bowel disease (IBD). The colitis that develops in these mice is associated with increased numbers of T helper cell (Th)2-type CD4 + TCR-ββ (CD4 + ββ) T cells producing predominantly interleukin (IL)-4. To investigate the role of these Th2-type CD4 + ββ T cells, we treated TCR-α −/− mice with anti–IL-4 monoclonal antibody (mAb). Approximately 60% of TCR-α −/− mice, including those treated with mock Ab and those left untreated, spontaneously developed IBD. However, anti–IL-4 mAb–treated mice exhibited no clinical or histological signs of IBD, and their levels of mucosal and systemic Ab responses were lower than those of mock Ab–treated mice. Although TCR-α −/− mice treated with either specific or mock Ab developed CD4 + ββ T cells, only those treated with anti–IL-4 mAb showed a decrease in Th2-type cytokine production at the level of mRNA and protein and an increase in interferon γ–specific expression. These findings suggest that IL-4–producing Th2-type CD4 + ββ T cells play a major immunopathological role in the induction of IBD in TCR-α −/− mice, a role that anti–IL-4 mAb inhibits by causing Th2-type CD4 + ββ T cells to shift to the Th1 type. inflammatory bowel disease T cell receptor α chain–deficient mice interleukin 4 mucosal immunity pathogenic T cell Th2-induced colitis Footnotes 1used in this paper: CD4+βdim, CD4+TCR-α−/β+; CD4+ββ, CD4+TCR-ββ; ELISPOT, enzyme-linked immunospot; IBD, inflammatory bowel disease; LP, lamina propria; MLN, mesenteric lymph node; RT, reverse-transcription; SP, spleen Submitted: 13 August 1998 Revision requested 21 June 1999 Accepted: 24 June 1999

Nishimura, Takashi; Iwakabe, Kenji; Sekimoto, Masashi; Ohmi, Yasushi; Yahata, Takashi; Nakui, Minoru; Sato, Takehito; Habu, Sonoko; Tashiro, Hiroyuki; Sato, Marimo; Ohta, Akio

doi: 10.1084/jem.190.5.617pmid: 10477547

The role of T helper type 1 (Th1) and Th2 cells in tumor immunity was investigated using Th cells induced from ovalbumin (OVA)-specific T cell receptor transgenic mice. Although Th1 cells exhibited stronger cytotoxicity than Th2 cells, both cell types completely eradicated tumors when transferred into mice bearing A20 tumor cells transfected with the OVA gene (A20-OVA). Th1 cells eradicated the tumor mass by inducing cellular immunity, whereas Th2 cells destroyed the tumor by inducing tumor necrosis. Both Th1 and Th2 cells required CD8 + T cells to eliminate tumors, and neither of these cells were able to completely eliminate A20-OVA tumors from T and B cell–deficient RAG2 −/− mice. Mice cured from tumors by Th1 and Th2 cell therapy rejected A20-OVA upon rechallenge, but CD8 + cytotoxic T lymphocytes were induced only from spleen cells prepared from cured mice by Th1 cell therapy. Moreover, we demonstrated that Th1 and Th2 cells used distinct adhesion mechanisms during tumor eradication: the leukocyte function-associated antigen (LFA)-1–dependent cell–cell adhesion step was essential for Th1 cell therapy, but not for Th2 cell therapy. These findings demonstrated for the first time the distinct role of antigen-specific Th1 and Th2 cells during eradication of established tumors in vivo. Th1 Th2 tumor adoptive immunotherapy cytokine Footnotes 1used in this paper: ICAM, intercellular adhesion molecule; OVA-pep, I-Ad–binding OVA323–339 peptide; RAG, recombination activating gene; Tg, transgenic; TRA, tumor-rejection antigen Submitted: 23 March 1999 Revision requested 23 June 1999 Accepted: 24 June 1999

Wu, Qiang; Wang, Yang; Wang, Jing; Hedgeman, Elizabeth O.; Browning, Jeffrey L.; Fu, Yang-Xin

doi: 10.1084/jem.190.5.629pmid: 10477548

Although several cytokines, including tumor necrosis factor (TNF), can promote the growth of dendritic cells (DCs) in vitro, the cytokines that naturally regulate DC development and function in vivo have not been well defined. Here, we report that membrane lymphotoxin (LT), instead of TNF, regulates the migration of DCs in the spleen. LTα −/− mice, lacking membrane LTα/β and LTα 3 , show markedly reduced numbers of DCs in the spleen. Unlike wild-type mice and TNF −/− mice that have densely clustered DCs in the T cell zone and around the marginal zone, splenic DCs in LTα −/− mice are randomly distributed. The reduced number of DCs in lymphoid tissues of LTα −/− mice is associated with an increased number of DCs in nonlymphoid tissues. The number of splenic DCs in LTα −/− mice is restored when additional LT-expressing cells are provided. Blocking membrane LTα/β in wild-type mice markedly diminishes the accumulation of DCs in lymphoid tissues. These data suggest that membrane LT is an essential ligand for the presence of DCs in the spleen. Mice deficient in TNF receptor, which is the receptor for both soluble LTα 3 and TNF-α 3 trimers, have normal numbers of DCs. However, LTβR −/− mice show reduced numbers of DCs, similar to the mice lacking membrane LT α/β. Taken together, these results support the notion that the signaling via LTβR by membrane LTα/β is required for the presence of DCs in lymphoid tissues. membrane lymphotoxin tumor necrosis factor dendritic cells lymphotoxin receptor migration Footnotes 1used in this paper: BM, bone marrow; DCs, dendritic cells; FDCs, follicular dendritic cells; LT, lymphotoxin; MLR, mixed leukocyte reaction; MZs, marginal zones; wt, wild-type Submitted: 26 March 1999 Revision requested 21 June 1999 Accepted: 22 June 1999

Wang, Haowei; Shlomchik, Mark J.

doi: 10.1084/jem.190.5.639pmid: 10477549

In systemic autoimmune disease, self-tolerance fails, leading to autoantibody production. A central issue in immunology is to understand the origins of activated self-reactive B cells. We have used immunoglobulin (Ig) transgenic mice to investigate the regulation of autoreactive B cells with specificity for self-IgG2a (the rheumatoid factor RF specificity) to understand how normal mice regulate RF autoantibodies and how this fails in autoimmune mice. We previously showed that normal mice do not tolerize the AM14 RF clone, nor do they appear to activate it. Here we show that in Fas-deficient autoimmune mice, the picture is quite different. RF B cells are activated to divide and secrete, but only when the autoantigen is present. Thus, B cells that are ignored rather than anergized in normal mice can be stimulated to produce autoantibody in Fas-deficient mice. This demonstrates a novel developmental step at which intact Fas–Fas ligand signaling is required to regulate B cells in order to prevent autoimmunity. These data also establish the relevance of ignorant self-specific B cells to autoantibody production in disease and prove that in the case of the RF specificity, the nominal autoantigen IgG2a is the driving autoantigen in vivo. autoimmunity systemic lupus B cell tolerance autoantibody IgG Footnotes 1used in this paper: AFCs, antibody-forming cells; ds, double-stranded; ELISpot, enzyme-linked immunospot assay; GC, germinal center; HEL, hen egg lysozyme; N, nontransgenic; RF, rheumatoid factor; ss, single-stranded; Tg, transgene; Tgic, transgenic Submitted: 7 April 1999 Revision requested 11 June 1999 Accepted: 28 June 1999

Anichini, Andrea; Molla, Alessandra; Mortarini, Roberta; Tragni, Gabrina; Bersani, Ilaria; Di Nicola, Massimo; Gianni, Alessandro M.; Pilotti, Silvana; Dunbar, Rod; Cerundolo, Vincenzo; Parmiani, Giorgio

doi: 10.1084/jem.190.5.651pmid: 10477550

It is not known if immune response to T cell–defined human histocompatibility leukocyte antigen (HLA) class I–restricted melanoma antigens leads to an expanded peripheral pool of T cells in all patients, affects cytotoxic T lymphocyte (CTL) generation, and correlates with anti-tumor response in metastatic lesions. To this end, a limiting dilution analysis technique was developed that allowed us to evaluate the same frequency of peptide-specific T cells as by staining T cells with HLA–peptide tetrameric complexes. In four out of nine patients, Melan-A/Mart-1 27–35 –specific CTL precursors (CTLp) were ≥1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO + memory T cell subset. In the remaining five patients, a low (<1/40,000) peptide-specific CTLp frequency was measured, and the precursors were only in the CD45RA + naive T cell subset. Evaluation of CTL effector frequency after bulk culture indicated that peptide-specific CTLs could be activated in all patients by using professional antigen-presenting cells as dendritic cells, but CTLp frequency determined the kinetics of generation of specificity and the final number of effectors as evaluated by both limiting dilution analysis and staining with HLA-A*0201–Melan-A/Mart-1 tetrameric complexes. Immunohistochemical analysis of 26 neoplastic lesions from the nine patients indicated absence of tumor regression in most instances, even in patients with an expanded peripheral T cell pool to Melan-A/Mart-1 and whose neoplastic lesions contained a high frequency of tetramer-positive Melan-A/Mart-1–specific T cells. Furthermore, frequent lack of a “brisk” or “nonbrisk” CD3 + CD8 + T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances. Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell–mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance. melanoma cytotoxic T lymphocytes Melan-A/Mart-1 peptide-specific CTL precursors tumor escape Footnotes 1used in this paper: DCs, dendritic cells; LDA, limiting dilution analysis; p, precursor; TILs, tumor-infiltrating lymphocytes; VGP, vertical growth phase Submitted: 15 February 1999 Revision requested 17 June 1999 Accepted: 21 June 1999

Baker, Jeanne E.; Kang, Joonsoo; Xiong, Na; Chen, Tempe; Cado, Dragana; Raulet, David H.

doi: 10.1084/jem.190.5.669pmid: 10477551

Transgenic expression constructs were employed to identify a cis-acting transcription element in the T cell receptor (TCR)-γ locus, called HsA, between the Vγ5 and Vγ2 genes. In constructs lacking the previously defined enhancer (3′E Cγ1 ), HsA supports transcription in mature but not immature T cells in a largely position-independent fashion. 3′E Cγ1 , without HsA, supports transcription in immature and mature T cells but is subject to severe position effects. Together, the two elements support expression in immature and mature T cells in a copy number–dependent, position-independent fashion. Furthermore, HsA was necessary for consistent rearrangement of transgenic recombination substrates. These data suggest that HsA provides chromatin-opening activity and, together with 3′E Cγ1 , constitutes a T cell–specific locus control region for the TCR-γ locus. T cell receptor γ locus control region V(D)J recombination transcription enhancer Footnotes 1used in this paper: DN, double-negative; DP, double-positive; LCR, locus control region; rr, rearranged; RT, reverse transcriptase; SP, single-positive Submitted: 17 March 1999 Revision requested 21 June 1999 Accepted: 28 June 1999

Kim, Chang H.; Qu, Cheng-Kui; Hangoc, Giao; Cooper, Scott; Anzai, Naoyuki; Feng, Gen-Sheng; Broxmeyer, Hal E.

doi: 10.1084/jem.190.5.681pmid: 10477552

Chemokines regulate a number of biological processes, including trafficking of diverse leukocytes and proliferation of myeloid progenitor cells. SHP-1 (Src homology 2 domain tyrosine phosphatase 1), a phosphotyrosine phosphatase, is considered an important regulator of signaling for a number of cytokine receptors. Since specific tyrosine phosphorylation of proteins is important for biological activities induced by chemokines, we examined the role of SHP-1 in functions of chemokines using viable motheaten (me v /me v ) mice that were deficient in SHP-1. Chemotactic responses to stromal call–derived factor 1 (SDF-1), a CXC chemokine, were enhanced with bone marrow myeloid progenitor cells as well as macrophages, T cells, and B cells from me v /me v versus wild-type (+/+) mice. SDF-1–dependent actin polymerization and activation of mitogen-activated protein kinases were also greater in me v /me v versus +/+ cells. In contrast, immature subsets of me v /me v bone marrow myeloid progenitors were resistant to effects of a number of chemokines that suppressed proliferation of +/+ progenitors. These altered chemokine responses did not appear to be due to enhanced expression of CXCR4 or lack of chemokine receptor expression. However, expression of some chemokine receptors (CCR1, CCR2, CCR3, and CXCR2) was significantly enhanced in me v /me v T cells. Our results implicate SHP-1 involvement in a number of different chemokine-induced biological activities. chemokine Src homology 2 domain tyrosine phosphatase 1 viable motheaten mice chemotaxis myelosuppression stromal cell–derived factor 1 Footnotes 1used in this paper: CFU-GM, colony forming unit-granulocyte and macrophage; HPC, hematopoietic progenitor cell; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; me, motheaten; mev, viable motheaten; SDF-1, stromal cell–derived factor 1; SHP-1, Src homology 2 domain tyrosine phosphatase 1 Submitted: 23 March 1999 Revision requested 27 May 1999 Accepted: 1 July 1999

Brard, Frederic; Shannon, Michele; Prak, Eline Luning; Litwin, Samuel; Weigert, Martin

doi: 10.1084/jem.190.5.691pmid: 10477553

Antibodies to single-stranded (ss)DNA are expressed in patients with systemic lupus erythematosus and in lupus-prone mouse models such as the MRL/Mp- lpr/lpr (MRL/ lpr ) strain. In nonautoimmune mice, B cells bearing immunoglobulin site-directed transgenes (sd-tgs) that code for anti-ssDNA are functionally silenced. In MRL/ lpr autoimmune mice, the same sd-tgs are expressed in peripheral B cells and these autoantibodies gain the ability to bind other autoantigens such as double-stranded DNA and cell nuclei. These new specificities arise by somatic mutation of the anti-ssDNA sd-tgs and by secondary light chain rearrangement. Thus, B cells that in normal mice are anergic can be activated in MRL/ lpr mice, which can lead to the generation of pathologic autoantibodies. In this paper, we provide the first direct evidence for peripheral rearrangement in vivo. anti-DNA B cell tolerance receptor editing systemic lupus erythematosus V H replacement Footnotes 1used in this paper: ANA, antinuclear antibody; dsDNA, double-stranded DNA; FW, framework; HEL, hen egg lysozyme; MRL/lpr, MRL/Mp-lpr/lpr mouse; RAG, recombination-activating gene; sd-tg, site-directed transgene; ssDNA, single-stranded DNA Submitted: 29 March 1999 Revision requested 23 June 1999 Accepted: 28 June 1999