High Frequencies of Naive Melan-a/Mart-1–Specific Cd8+ T Cells in a Large Proportion of Human Histocompatibility Leukocyte Antigen (Hla)-A2 IndividualsPittet, Mikaël J.; Valmori, Danila; Dunbar, P. Rod; Speiser, Daniel E.; Liénard, Danielle; Lejeune, Ferdy; Fleischhauer, Katharina; Cerundolo, Vincenzo; Cerottini, Jean-Charles; Romero, Pedro
doi: 10.1084/jem.190.5.705pmid: 10477554
Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A–specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A–specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (≥1 in 2,500 CD8 + T cells) of Melan-A–specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A–specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RA hi /RO − phenotype, whereas variable proportions of Ag-experienced CD45RA lo /RO + Melan-A–specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix–specific CTLs from all individuals exhibited a CD45RA lo /RO + memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A + cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon γ ELISPOT assays independently confirmed that most of the Melan-A–specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A–specific CD8 + T cells can be found in a large proportion of HLA-A*0201 + individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A–specific cells can occur in vivo. melanoma tetramer influenza matrix immunotherapy tumor immunity Footnotes 1used in this paper: CTL, cytolytic T lymphocyte; CTLp, cytolytic T lymphocytes precursor(s); LDA, limiting dilution analysis; Melan-A, Melan-A/MART-1 Submitted: 1 April 1999 Revision requested 30 June 1999 Accepted: 6 July 1999
Mycobacterium tuberculosis Expresses a Novel Ph-Dependent Divalent Cation Transporter Belonging to the Nramp FamilyAgranoff, Daniel; Monahan, Irene M.; Mangan, Joseph A.; Butcher, Philip D.; Krishna, Sanjeev
doi: 10.1084/jem.190.5.717pmid: 10477555
Mammalian natural resistance–associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria. Eukaryotic Nramp homologues transport divalent cations such as Fe 2+ , Mn 2+ , Zn 2+ , and Cu 2+ . Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guérin BCG) also encode an Nramp homologue (Mramp). RNA encoding Mramp induces ∼20-fold increases in 65 Zn 2+ and 55 Fe 2+ uptake when injected into Xenopus laevis oocytes. Transport is dependent on acidic extracellular pH and is maximal between pH 5.5 and 6.5. Mramp-mediated 65 Zn 2+ and 55 Fe 2+ transport is abolished by an excess of Mn 2+ and Cu 2+ , confirming that Mramp interacts with a broad range of divalent transition metal cations. Using semiquantitative reverse transcription PCR, we show that Mramp mRNA levels in M. tuberculosis are upregulated in response to increases in ambient Fe 2+ and Cu 2+ between <1 and 5 μM concentrations and that this upregulation occurs in parallel with mRNA for y39, a putative metal-transporting P-type ATPase. Using a quantitative ratiometric PCR technique, we demonstrate a fourfold decrease in Mramp/y39 mRNA ratios from organisms grown in 5–70 μM Cu 2+ . M. bovis BCG cultured axenically and within THP-1 cells also expresses mRNA encoding Mramp. Mramp exemplifies a novel prokaryotic class of metal ion transporter. Within phagosomes, Mramp and Nramp1 may compete for the same divalent cations, with implications for intracellular survival of mycobacteria. bacillus Calmette-Guérin Xenopus oocyte metal ion phagosome intracellular pathogen Footnotes 1used in this paper: BCG, bacillus Calmette-Guérin; 2-DOG, 2′-deoxy-14Cd-glucose; Mramp, mycobacterial homologue of Nramp; Nramp, natural resistance–associated macrophage protein; ORF, open reading frame; RT, reverse transcription; SUM, standard uptake medium; TC, tandem competitive Submitted: 31 March 1999 Revision requested 2 June 1999 Accepted: 6 July 1999
Studies in B7-Deficient Mice Reveal a Critical Role for B7 Costimulation in Both Induction and Effector Phases of Experimental Autoimmune EncephalomyelitisChang, Tammy T.; Jabs, Claudia; Sobel, Raymond A.; Kuchroo, Vijay K.; Sharpe, Arlene H.
doi: 10.1084/jem.190.5.733pmid: 10477557
The importance of B7 costimulation in regulating T cell expansion and peripheral tolerance suggests that it may also play a significant regulatory role in the development of autoimmune disease. It is unclear whether B7 costimulation is involved only in the expansion of autoreactive T cells in the periphery, or if it is also required for effector activation of autoreactive T cells in the target organ for mediating tissue injury and propagating autoimmune disease. In this study, the role of B7–CD28 costimulation and the relative importance of B7 costimulators for the induction and effector phases of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide were examined. Wild-type, B7-1/B7-2–deficient mice, or CD28-deficient C57BL/6 mice were immunized with MOG 35-55 peptide. Mice lacking both B7-1 and B7-2 or CD28 showed no or minimal clinical signs of EAE and markedly reduced inflammatory infiltrates in the brain and spinal cord. However, mice lacking either B7-1 or B7-2 alone developed clinical and pathologic EAE that was comparable to EAE in wild-type mice, indicating overlapping functions for B7-1 and B7-2. Resistance to EAE was not due to a lack of induction of T helper type 1 (Th1) cytokines, since T cells from B7-1/B7-2 −/− mice show reduced proliferative responses, but greater interferon γ production compared with T cells from wild-type mice. To study the role of B7 molecules in the effector phase of the disease, MOG 35-55–specific T lines were adoptively transferred into the B7-1/B7-2 −/− and wild-type mice. Clinical and histologic EAE were markedly reduced in B7-1/B7-2 −/− compared with wild-type recipient mice. These results demonstrate that B7 costimulation has critical roles not only in the initial activation and expansion of MOG-reactive T cells, but also in the effector phase of encephalitogenic T cell activation within the central nervous system. B7 costimulation knockout mouse autoimmunity experimental autoimmune encephalomyelitis Footnotes 1used in this paper: CNS, central nervous system; CTLA-4, CTL-associated molecule 4; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein Submitted: 15 June 1999 Accepted: 29 June 1999
N-Formylpeptides Induce Two Distinct Concentration Optima for Mouse Neutrophil Chemotaxis by Differential Interaction with Two N-Formylpeptide Receptor (Fpr) SubtypesHartt, Jennifer K.; Barish, Grant; Murphy, Philip M.; Gao, Ji-Liang
doi: 10.1084/jem.190.5.741pmid: 10477558
The N -formylpeptide receptor (FPR) is a G protein–coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N -formylpeptides. Here we show that a mouse gene named Fpr-rs2 encodes a second N -formylpeptide receptor subtype selective for neutrophils which we have provisionally named FPR2. The prototype N -formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2. The EC 50 s, ∼5 μM for calcium flux and chemotaxis, were ∼100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells. Consistent with this, fMLF induced two distinct concentration optima for chemotaxis of normal mouse neutrophils, but only the high concentration optimum for chemotaxis of neutrophils from FPR knockout mice. Based on these data, we hypothesize that high- and low-affinity N -formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N -formylpeptide gradients. chemoattractant inflammation neutrophil G protein–coupled receptor phagocyte Footnotes G. Barish's present address is University of Michigan School of Medicine, Ann Arbor, MI 48105. J.K. Hartt's present address is Immunology Department, Harvard Medical School, 200 Longwood Ave., Bldg. D-2, Rm. 137, Boston, MA 02115. Submitted: 29 March 1999 Revision requested 25 June 1999 Accepted: 6 July 1999