David J. Topham

Jak2 Is Essential for Signaling through a Variety of Cytokine Receptors

A variety of cytokines activate receptor-associated members of the Janus family of protein tyrosine kinases (Jaks). To assess the role of Jak2, we have derived Jak2-deficient mice. The mutation causes an embryonic lethality due to the absence of definitive erythropoiesis. Fetal liver myeloid progenitors, although present based on the expression of lineage specific markers, fail to respond to erythropoietin, thrombopoietin, interleukin-3 (IL-3), or granulocyte/macrophage colony-stimulating factor. In contrast, the response to granulocyte specific colony-stimulating factor is unaffected. Jak2-deficient fibroblasts failed to respond to interferon gamma (IFNgamma), although the responses to IFNalpha/beta and IL-6 were unaffected. Lastly, reconstitution experiments demonstrate that Jak2 is not required for the generation of lymphoid progenitors, their amplification, or functional differentiation. Therefore, Jak2 plays a critical, nonredundant role in the function of a specific group of cytokines receptors.

Stat5 Is Required for IL-2-Induced Cell Cycle Progression of Peripheral T Cells

Many cytokines activate two highly homologous Stat proteins, 5a and 5b. Mice deficient in both genes lack all growth hormone and prolactin functions but retain functions associated with cytokines such as erythropoietin. Here, we demonstrate that, while lymphoid development is normal, Stat5a/b mutant peripheral T cells are profoundly deficient in proliferation and fail to undergo cell cycle progression or to express genes controlling cell cycle progression. In addition, the mice lack NK cells, develop splenomegaly, and have T cells with an activated phenotype, phenotypes seen in IL-2 receptor beta chain-deficient mice. These phenotypes are not seen in mice lacking Stat5a or Stat5b alone. The results demonstrate that the Stat5 proteins, redundantly, are essential mediators of IL-2 signaling in T cells.

SOCS1 Deficiency Causes a Lymphocyte-Dependent Perinatal Lethality

SOCS1 is an SH2-containing protein that is primarily expressed in thymocytes in a cytokine- and T cell receptor-independent manner. SOCS1 deletion causes perinatal lethality with death by 2-3 weeks. During this period thymic changes include a loss of cellularity and a switch from predominantly CD4+ CD8+ to single positive cells. Peripheral T cells express activation antigens and proliferate to IL-2 in the absence of anti-CD3. In addition, IFNgamma is present in the serum. Reconstitution of the lymphoid lineage of JAK3-deficient mice with SOCS1-deficient stem cells recapitulates the lethality and T cell alterations. Introducing a RAG2 or IFNgamma deficiency eliminates lethality. The results demonstrate that lymphocytes are critical to SOCS1-associated perinatal lethality and implicate SOCS1 in lymphocyte differentiation or regulation.

Effector CD4+ and CD8+ T-cell mechanisms in the control of respiratory virus infections.

The rules for T‐cell‐mediated control of viruses that infect via the respiratory mucosae show both common themes and differences depending on the nature of the pathogen. Virus‐specific CD8+ cytotoxic T lymphocytes (CTLs) are the key effectors of virus clearance in mice infected with both negative strand RNA viruses (influenza and Sendai) and a DNA virus, the murine γ‐herpesvirus68 (MHV‐68). Recently completed experiments establish that these activated CD8+ T cells indeed operate primarily via contact‐dependent lysis, Perform‐mediated cytotoxicity seems to be the preferred mode, though a Fas‐based mechanism can apparently serve as an alternative mechanism. Immune CD4+ T cells functioning in the absence of the CD8+ subset cannot eliminate MHV‐68 from lung epithelial cells, are somewhat less efficient than the CD8+ CTLs at clearing the RNA viruses, and are generally ineffectual in mice that lack B lymphocytes. Though cytokine secretion by CD4+ and CD8+ T cells in the virus‐infected king may promote both T‐cell extravasation and macrophage activation, such processes are not alone sufficient to deal consistently with any d these infections. However, CD4+ T help is mandatory for an effective B‐cell response, and can operate lo promote the clonal expansion of virus‐specific CD8+ T cells in the lymph nodes and spleen. Furthermore, a concurrent CD4+ T‐cell response seems to be essential for maintaining continued CD8+ T‐cell surveillance and effector capacity through the persistent, latent phase of MHV‐68 infection in B cells. Thus, the evidence to date supports a very traditional view: CD8+ T cells function mainly as killers and the CD4+ T cells as helpers in these respiratory virus infections.

SOCS3 Is Essential in the Regulation of Fetal Liver Erythropoiesis

SOCS3 (CIS3/JAB2) is an SH2-containing protein that binds to the activation loop of Janus kinases, inhibiting kinase activity, and thereby suppressing cytokine signaling. During embryonic development, SOCS3 is highly expressed in erythroid lineage cells and is Epo independent. Transgene-mediated expression blocks fetal erythropoiesis, resulting in embryonic lethality. SOCS3 deletion results in an embryonic lethality at 12-16 days associated with marked erythrocytosis. Moreover, the in vitro proliferative capacity of progenitors is greatly increased. SOCS3-deficient fetal liver stem cells can reconstitute hematopoiesis in lethally irradiated adults, indicating that its absence does not disturb bone marrow erythropoiesis. Reconstitution of lymphoid lineages in JAK3-deficient mice also occurs normally. The results demonstrate that SOCS3 is critical in negatively regulating fetal liver hematopoiesis.

The Collagen Binding α1β1 Integrin VLA-1 Regulates CD8 T Cell-Mediated Immune Protection against Heterologous Influenza Infection

A common feature of many infections is that many pathogen-specific memory T cells become established in diverse nonlymphoid tissues. A mechanism that promotes the retention and survival of the memory T cells in diverse tissues has not been described. Our studies show that the collagen binding α1β1 integrin, VLA-1, is expressed by the majority of influenza-specific CD8 T cells recovered from nonlymphoid tissues during both the acute and memory phases of the response. Antibody treatment or genetic deficiency of VLA-1 decreased virus-specific CTL in the lung and other nonlymphoid tissues, and increased them in the spleen. In spite of the increase in the spleen, secondary heterosubtypic immunity against flu was compromised. This suggests that VLA-1 is responsible for retaining protective memory CD8 T cells in the lung and other tissues via attachment to the extracellular matrix.

Stat5 Activation Is Uniquely Associated with Cytokine Signaling in Peripheral T Cells

The activation and subsequent proliferation of peripheral T cells requires the engagement of the T cell and a cytokine receptor, typically the IL-2 or IL-4 receptors. Critical to understanding the regulation of peripheral T cells is the knowledge of the unique contributions of each receptor to full T cell activation and cell cycle progression. Mice deficient in Stat5a and Stat5b have demonstrated the essential role that these highly related proteins play in cell cycle progression following peripheral T cell activation. Here we demonstrate that activation of the Stat5 proteins by tyrosine phosphorylation is uniquely contributed by cytokine receptor signaling and specifically does not occur through the T cell receptor complex.

The Role of Antigen in the Localization of Naive, Acutely Activated, and Memory CD8+ T Cells to the Lung During Influenza Pneumonia

The role of Ag in the recruitment and localization of naive, acutely activated, and memory CD8+ T cells to the lung during influenza infection was explored using TCR-transgenic (Tg) mice. Naive, Thy1.2+CD8+ OT-I TCR-Tg cells were primed and recruited to the lung after transfer into congenic Thy1.1+ recipients challenged with a genetically engineered influenza virus (influenza A/WSN/33 (WSN)-OVAI) containing the Kb restricted OVA257–264 epitope (siinfekl) in the viral neuraminidase stalk. However, if the transferred animals were infected with a similar influenza virus that expressed an irrelevant Kb epitope (WSN-PEPII), no TCR-Tg T cells were detectable in the lung, although they were easily visible in the lymphoid organs. Conversely, there were substantial numbers of OT-I cells found in the lungs of WSN-PEPII-infected mice when the animals had been previously, or were concurrently, infected with a recombinant vaccinia virus expressing OVA. Similar results were obtained with nontransgenic populations of memory CD8+ T cells reactive to a murine γ-herpesvirus-68 Ag. Interestingly, the primary host response to the immunodominant influenza nucleoprotein epitope was not affected by the presence of memory or recently activated OT-I T cells. Thus, although Ag is required to activate the T cells, the subsequent localization of T cells to the lung during a virus infection is a property of recently activated and memory T cells and is not necessarily driven by Ag in the lung.

Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice.

Inherited deficiency of the CD40 ligand (X-linked hyper-IgM syndrome) is characterized by failure of immunoglobulin isotype switching and severe defects of cell-mediated immunity. To test the potential for gene transfer therapy to correct this disorder, we transduced murine bone marrow or thymic cells with a retroviral vector containing the cDNA for the murine CD40 ligand (CD40L) and injected them into CD40L–/– mice. Even low-level, constitutive expression of the transgene stimulated humoral and cellular immune functions in these mice. With extended follow-up, however, 12 of 19 treated mice developed T-lymphoproliferative disorders, ranging from polyclonal increases of lymphoblasts to overt monoclonal T-Lymphoblastic lymphomalymphomas that involved multiple organs. Our findings show that constitutive (rather than tightly regulated), low-level expression of CD40L can produce abnormal proliferative responses in developing T lymphocytes, apparently through aberrant interaction between CD40L+ and TCRαβ+CD40+ thymocytes. Current methods of gene therapy may prove inappropriate for disorders involving highly regulated genes in essential positions in proliferative cascades.

Quantifying the Early Immune Response and Adaptive Immune Response Kinetics in Mice Infected with Influenza A Virus

ABSTRACT Seasonal and pandemic influenza A virus (IAV) continues to be a public health threat. However, we lack a detailed and quantitative understanding of the immune response kinetics to IAV infection and which biological parameters most strongly influence infection outcomes. To address these issues, we use modeling approaches combined with experimental data to quantitatively investigate the innate and adaptive immune responses to primary IAV infection. Mathematical models were developed to describe the dynamic interactions between target (epithelial) cells, influenza virus, cytotoxic T lymphocytes (CTLs), and virus-specific IgG and IgM. IAV and immune kinetic parameters were estimated by fitting models to a large data set obtained from primary H3N2 IAV infection of 340 mice. Prior to a detectable virus-specific immune response (before day 5), the estimated half-life of infected epithelial cells is ∼1.2 days, and the half-life of free infectious IAV is ∼4 h. During the adaptive immune response (after day 5), the average half-life of infected epithelial cells is ∼0.5 days, and the average half-life of free infectious virus is ∼1.8 min. During the adaptive phase, model fitting confirms that CD8+ CTLs are crucial for limiting infected cells, while virus-specific IgM regulates free IAV levels. This may imply that CD4 T cells and class-switched IgG antibodies are more relevant for generating IAV-specific memory and preventing future infection via a more rapid secondary immune response. Also, simulation studies were performed to understand the relative contributions of biological parameters to IAV clearance. This study provides a basis to better understand and predict influenza virus immunity.