Coronavirus disease 2019 subphenotypes and differential treatment response to convalescent plasma in critically ill adults: secondary analyses of a randomized clinical trial.

PURPOSE: Benefit from convalescent plasma therapy for coronavirus disease 2019 (COVID-19) has been inconsistent in randomized clinical trials (RCTs) involving critically ill patients. As COVID-19 patients are immunologically heterogeneous, we hypothesized that immunologically similar COVID-19 subphenotypes may differ in their treatment responses to convalescent plasma and explain inconsistent findings between RCTs . METHODS: We tested this hypothesis in a substudy involving 1239 patients, by measuring 26 biomarkers (cytokines, chemokines, endothelial biomarkers) within the randomized, embedded, multifactorial, adaptive platform trial for community-acquired pneumonia (REMAP-CAP) that assigned 2097 critically ill COVID-19 patients to either high-titer convalescent plasma or usual care. Primary outcome was organ support free days at 21 days (OSFD-21) . RESULTS: Unsupervised analyses identified three subphenotypes/endotypes. In contrast to the more homogeneous subphenotype-2 (N = 128 patients, 10.3%; with elevated type i and type ii effector immune responses) and subphenotype-3 (N = 241, 19.5%; with exaggerated inflammation), the subphenotype-1 had variable biomarker patterns (N = 870 patients, 70.2%). Subphenotypes-2, and -3 had worse outcomes, and subphenotype-1 had better outcomes with convalescent plasma therapy compared with usual care (median (IQR). OSFD-21 in convalescent plasma vs usual care was 0 (- 1, 21) vs 10 (- 1, to 21) in subphenotype-2; 1.5 (- 1, 21) vs 12 (- 1, to 21) in suphenotype-3, and 0 (- 1, 21) vs 0 (- 1, to 21) in subphenotype-1 (test for between-subphenotype differences in treatment effects p = 0.008). CONCLUSIONS: We reported three COVID-19 subphenotypes, among critically ill adults, with differential treatment effects to ABO-compatible convalescent plasma therapy. Differences in subphenotype prevalence between RCT populations probably explain inconsistent results with COVID-19 immunotherapies.

Genetic transformation of Dichanthium annulatum (Forssk)--an apomictic tropical forage grass.

Eleven Dichanthium annulatum (Forssk) plants were regenerated from embryogenic callus co-transformed with two plasmids encoding either the hygromycin phosphotransferase gene (hph) or the beta-glucuronidase (GUS) gene (uidA). Analysis of these putative transformants showed that three plants were transformed with the hph gene, showed the presence of the hph transcript and expressed hygromycin resistance after transfer to soil. Two of these also contained the uidA gene but did not express GUS and were shown to be the same transformation event. All three of the transformants set seed. Hygromycin resistance varied from 68-100% in the progeny of the three transformants. Transgene transmission appeared to have been mainly through apomixis.

Agrobacterium tumefaciens-mediated transformation of Festuca arundinacea (Schreb.) and Lolium multiflorum (Lam.).

Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and beta-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and surviving colonies plated on embryogenesis media. Eight Lolium (six independent lines) and two Festuca plants (independent lines) were regenerated and established in soil. All plants were hygromycin-resistant, but histochemical determination of GUS activity showed that only one Festuca plant and one Lolium plant expressed GUS. Three GUS-negative transgenic L. multiflorum and the two F. arundinacea plants were vernalised and allowed to flower. All three Lolium plants were male- and female-fertile, but the Festuca plants failed to produce seed. Progeny analysis of L. multiflorum showed a 24-68% inheritance of the hph and uidA genes in the three lines with no significant difference between paternal and maternal gene transmission. However, significant differences were noted between the paternal and maternal expression of hygromycin resistance.

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.

The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyer's patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

Receptor-specific allelic exclusion of TCRV alpha-chains during development.

Expression of a single Ag receptor on lymphocytes is maintained via allelic exclusion that generates cells with a clonal receptor repertoire. We show in normal mice and mice expressing functionally rearranged TCR alphabeta transgenes that allelic exclusion at the TCR alpha locus is not operational in immature thymocytes, whereas most mature T cells express a single TCRV alpha-chain. TCRV alpha allelic exclusion in mature thymocytes is regulated through a CD45 tyrosine phosphatase-mediated signal during positive selection. Using functional and genetic systems for selection of immature double TCRV alpha+ thymocytes, we show that peptide-specific ligand recognition provides the signal for allelic exclusion, i.e., mature T cells maintain expression of the ligand-specific TCRV alpha-chain, but lose the nonfunctional receptor. Whereas activation of TCRV beta-chains or CD3epsilon leads to receptor internalization, TCRV alpha ligation promotes retention of the TCR on the cell surface. Although both TCRV alpha- and TCRV beta-chains trigger phosphotyrosine signaling, only the TCRV beta-chain mediates membrane recruitment of the GTPase dynamin. These data indicate that TCRV alpha-directed signals for positive selection control allelic exclusion in T cells, and that developmental signals can select for single receptor usage.

Effective and long-lasting immunity against the parasite Leishmania major in CD8-deficient mice.

The results of earlier investigations that tested whether CD8(+) T cells are required in the defense against Leishmania major have been inconsistent. We used CD8-deficient mice to directly address this issue. After primary infection with L. major, CD8-deficient mice controlled the infection for over 1 year and mounted strong T helper 1 cell responses. Thus, CD8(+) T cells are not required for the long-term control of a primary infection with L. major.

Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.

The ligand for osteoprotegerin has been identified, and it is a TNF-related cytokine that replaces the requirement for stromal cells, vitamin D3, and glucocorticoids in the coculture model of in vitro osteoclastogenesis. OPG ligand (OPGL) binds to a unique hematopoeitic progenitor cell that is committed to the osteoclast lineage and stimulates the rapid induction of genes that typify osteoclast development. OPGL directly activates isolated mature osteoclasts in vitro, and short-term administration into normal adult mice results in osteoclast activation associated with systemic hypercalcemia. These data suggest that OPGL is an osteoclast differentiation and activation factor. The effects of OPGL are blocked in vitro and in vivo by OPG, suggesting that OPGL and OPG are key extracellular regulators of osteoclast development.

The interferon regulatory transcription factor IRF-1 controls positive and negative selection of CD8+ thymocytes.

Little is known about the molecular mechanisms and transcriptional regulation that govern T cell selection processes and the differentiation of CD4+ and CD8+ T cells. Mice lacking the interferon regulatory transcription factor-1 (IRF-1) have reduced numbers of mature CD8+ cells within the thymus and peripheral lymphatic organs. Here we show that positive and negative T cell selection of two MHC class I-restricted TCR alphabeta transgenes, H-Y and P14, are impaired in IRF-1-/- mice. The absence of IRF-1 resulted in decreased expression of LMP2, TAP1, and MHC class I on thymic stromal cells. Despite decreased MHC class I expression on IRF-1-/- thymic stromal cells, the defect in CD8+ T cells development did not reside in the thymic environment, and IRF-1-/- stromal cells can fully support development of CD8+ thymocytes in in vivo bone marrow chimeras and in vitro reaggregation cultures. Moreover, IRF-1-/- thymocytes displayed impaired TCR-mediated signal transduction, and the induction of negative selection in TCR Tg thymocytes from IRF-1-/- mice required a 1000-fold increase in selecting peptide. We also provide evidence that IRF-1 is mainly expressed in mature, but not immature, thymocytes and that expression of IRF-1 in immature thymocytes is induced after peptide-specific TCR activation. These results indicate that IRF-1 regulates gene expression in developing thymocytes required for lineage commitment and selection of CD8+ thymocytes.

T cell development in mice expressing splice variants of the protein tyrosine phosphatase CD45.

The transmembrane protein tyrosine phosphatase CD45 is expressed in multiple isoforms as a result of alternative splicing of variable exons encoding the extracellular domain. CD45 expression is critical for T cell development, and thymocyte maturation is blocked at the immature CD4+ CD8+ double-positive stage in CD45 gene-deficient (CD45 -/-) mice. Moreover, splicing of variable CD45 exons changes during thymocyte selection. To test the role of CD45 extracellular splice variants in T cell selection and development, we introduced CD45RO (a low-m.w. splice variant lacking exons 4, 5, and 6) and CD45ABC (a high-m.w. isoform containing all exons) transgenes under the control of a thymocyte-specific promoter into a CD45 -/- background, generating CD45RO transgene-positive CD45 -/- (CD45RO) and CD45ABC transgene-positive CD45 -/- (CD45ABC) mice. We demonstrate that both CD45 splice isoforms can rescue development of CD4+ and CD8+ TCR-alphabeta+ thymocytes. Neither CD45 isoform rescued positive selection of H-Y TCR transgene thymocytes, and these cells were blocked at a HSA(high) CD69- CD5(low) stage of development. Peripheral T cells from CD45RO and CD45ABC mice proliferated in response to allogeneic stimulator cells and anti-CD3epsilon cross-linking. However, only CD45RO mice, not CD45ABC mice, generated cytotoxic T cell responses and neutralizing, Th cell-dependent IgG Abs after viral infections. In addition, we show that T cells from CD45RO and CD45ABC mice accumulate in lymph nodes but not in the spleen, liver, or skin, indicating that the CD45 phosphatase may control the homing behavior and trafficking of T cells.

Alterations in the level of CD45 surface expression affect the outcome of thymic selection.

CD45 is a receptor protein tyrosine phosphatase whose activity is required for thymocyte development and TCR-mediated signal transduction. Here we show that positive selection of TCR-alphabeta transgenic thymocytes is completely blocked in CD45 exon 6 -/- gene-deficient (CD45 -/-) mice that express the P14 TCR specific for the lymphocytic choriomeningitis virus. Thymocytes from mice heterozygous for the targeted disruption of the CD45 gene (CD45 +/-) displayed a reduction in both CD45 surface intensity and enzymatic CD45 protein tyrosine phosphatase activity. Surprisingly, positive thymocyte selection was enhanced in CD45 +/- mice as characterized by an up-regulation of the P14 TCR on thymocytes and increased numbers of transgenic T cells. Using a variant of lymphocytic choriomeningitis virus that is impaired in the induction of negative selection of P14 thymocytes, we also show that the reduction of CD45 surface expression in CD45 +/- mice rendered P14 transgene thymocytes susceptible to negative selection. These data demonstrate that changes in the expression level of the receptor protein tyrosine phosphatase CD45 can alter thymic selection.

Comparison of ribosomal DNA sites in Lolium species by fluorescence in situ hybridization.

The position of the 18S-5.8S-26S and 5S rRNA genes have been physically mapped on the chromosomes of seven Lolium taxa. 18S-5.8S-26S sites were seen on two pairs of chromosomes in the inbreeding taxa. In the outbreeding taxa six sites were found in the L. multiflorum, seven in L. perenne and nine in L. rigidum var. rigidum. Two 5S sites were found in each of the taxa. In the inbreeders, the 5S sites were found adjacent to the 18S-5.8S-26S sites on chromosome 2. In L. multifiorum and L.perenne the 5S sites were on the short arm of chromosome 3. However, in L. rigidum var. rigidum the 5S rDNA site was found in either of the two positions.

Impaired negative selection of T cells in Hodgkin's disease antigen CD30-deficient mice.

CD30 is found on Reed-Sternberg cells of Hodgkin's disease and on a variety of non-Hodgkin's lymphoma cells and is up-regulated on cells after Epstein-Barr virus, human T cell leukemia virus, and HIV infections. We report here that the thymus in CD30-deficient mice contains elevated numbers of thymocytes. Activation-induced death of thymocytes after CD3 cross-linking is impaired both in vitro and in vivo. Breeding the CD30 mutation separately into alpha beta TCR-or gamma delta TCR-transgenic mice revealed a gross defect in negative but not positive selection. Thus, like TNF-receptors and Fas/Apo-1, the CD30 receptor is involved in cell death signaling. It is also an important coreceptor that participates in thymic deletion.

Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4.

The role of the cell-surface molecule CTLA-4 in the regulation of T cell activation has been controversial. Here, lymph nodes and spleens of CTLA-4-deficient mice accumulated T cell blasts with up-regulated activation markers. These blast cells also infiltrated liver, heart, lung, and pancreas tissue, and amounts of serum immunoglobulin were elevated. The mice invariably became moribund by 3 to 4 weeks of age. Although CTLA-4-deficient T cells proliferated spontaneously and strongly when stimulated through the T cell receptor, they were sensitive to cell death induced by cross-linking of the Fas receptor and by gamma irradiation. Thus, CTLA-4 acts as a negative regulator of T cell activation and is vital for the control of lymphocyte homeostasis.

Alloreactive gamma delta thymocytes utilize distinct costimulatory signals from peripheral T cells.

Interactions between CD28/CTLA-4 on T cells and CD80 (B7.1) and CD86 (B7.2) counter receptors provide crucial costimulatory signals for TCR-alpha beta+ lymphocytes. To test the role of CD28 in thymic development and activation of TCR-gamma delta+ T cells, we introduced the alloreactive V gamma 2V alpha 11.3 TCR into CD28-deficient mice (CD28-/-). We show that positive and negative selection of gamma delta Tg thymocytes proceeded normally in the absence of CD28. Although mature Tg gamma delta+ thymocytes required a second costimulatory signal for proliferation, gamma delta+ thymocytes from CD28-/- and CD28+/- littermates responded equally well to the alloantigen Tlab. Alloreactivity of CD28-/- and CD28+/- Tg gamma delta+ thymocytes could not be blocked with mAbs against CD80 and CD86 ligands. Thus gamma delta thymocytes utilize a costimulatory system during development and alloresponses that is independent of CD28/CD80 and CD28/CD86 interactions. By contrast to V gamma 2V alpha 11.3+ thymocytes, alloreactivity of V gamma 2V alpha 11.3+ lymph node T cells depended on CD28 costimulation and was severely impaired in CD28-/- mice. These data provide functional evidence that maturation and selection of gamma delta cells is independent of CD28. These results also indicate that distinct costimulatory pathways are operational in mature thymocytes and peripheral T cells.

T cell repertoire and clonal deletion of Mtv superantigen-reactive T cells in mice lacking CD4 and CD8 molecules.

CD4-CD8- double-negative T cells constitute a lymphocyte subpopulation within the thymus and peripheral lymphatic organs that express a unique T cell receptor (TCR) repertoire and do not undergo negative selection. To test whether these cells develop as a distinct lineage or due to altered selection in the absence of CD4 and CD8 expression, we analyzed the TCR repertoire in mice lacking both CD4 and CD8 accessory molecules after homologous recombination (CD40/0CD80/0). We show that mature T cells of CD40/0CD80/0 mice express an unbiased diverse TCR V beta repertoire comparable to wild type mice. In addition, clonal deletion of mouse mammary tumor virus superantigen-reactive T cells did occur in CD40/0CD80/0 mice. These data show that the intrinsic lack of CD4 and CD8 expression has no effect on the mature TCR repertoire and that clonal deletion of superantigen-reactive cells is independent of CD4 and CD8 co-receptors.

Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor beta.

In mice lacking the interleukin-2 receptor beta chain (IL-2R beta), T cells were shown to be spontaneously activated, resulting in exhaustive differentiation of B cells into plasma cells and the appearance of high serum concentrations of immunoglobulins G1 and E as well as autoantibodies that cause hemolytic anemia. Marked infiltrative granulocytopoiesis was also apparent, and the animals died after about 12 weeks. Depletion of CD4+ T cells in mutant mice rescued B cells without reversion of granulocyte abnormalities. T cells did not proliferate in response to polyclonal activators, nor could antigen-specific immune responses be elicited. Thus, IL-2R beta is required to keep the activation programs of T cells under control, to maintain homeostasis, and to prevent autoimmunity.

T lymphocyte development in p56lck deficient mice: allelic exclusion of the TcR beta locus is incomplete but thymocyte development is not restored by TcR beta or TcR alpha beta transgenes.

The protein tyrosine kinase, p56lck, is involved in signal transduction in mature T cells and in the molecular events controlling early thymocyte differentiation. Thymuses of mice deficient for p56lck expression (p56lck-/-) consist of immature CD4-CD8- double-negative (DN) and CD4+CD8+ double-positive (DP) thymocytes and are severely reduced in total cell number. In this report we have studied DN thymocytes from p56lck-/- mice and found an increase in the proportion of the CD44-CD25+ subset, suggesting that transit through this stage, which is known to require T cell receptor (TcR) beta expression, may be delayed in the absence of p56lck expression. In addition, the expression of a transgenic TcR beta chain or TcR alpha beta pair did not restore thymic development in p56lck-/- mice. However, in contrast to mice expressing a dominant negative isoform of p56lck in which DP thymocytes do not develop, DP thymocytes still develop in nontransgenic and TcR transgenic p56lck-/- mice. These results demonstrate that expansion of the DP subset is impaired in p56lck-/- mice. In contrast, allelic exclusion is not severely compromised. Although there was an increase in the number of peripheral T cells expressing more than one V beta chain in TcR transgenic p56lck-/- mice, we found that inhibition of endogenous TcR beta gene rearrangement was almost complete in thymocytes of V beta transgenic p56lck-/- mice and we could not detect any peripheral T cells that expressed more than one V beta chain in non-transgenic p56lck-/- mice.

Spontaneous resistance to acute T-cell leukaemias in TCRV gamma 1.1J gamma 4C gamma 4 transgenic mice.

The concept of tumour surveillance implies that specific and non-specific components of the immune system eliminate tumours in the early phase of malignancy. The immunological mechanisms that control growth of preneoplastic cells are, however, not known. T cells expressing gamma delta T-cell receptors (TCR) were first described as lymphocytes with reactivity against various tumour cells, which suggests that gamma delta T cells could mediate tumour surveillance. Here we show that TCRV gamma 1.1J gamma 4C gamma 4 transgenic mice are spontaneously resistant to acute T-cell leukaemias but cannot reject non-haematopoietic tumours. TCRV gamma 1.1J gamma 4C gamma 4+ hybridomas isolated from these mice react in vitro against almost all haematopoietic tumour cell lines tested. Recognition of tumour cells depends on the gamma delta TCR but is independent of major histocompatibility complex (MHC) class I, MHC class II, or TAP-2 peptide transporter expression. Ligand recognition is influenced by the murine Nromp gene, which confers resistance or susceptibility to tuberculosis, lepra and leishmaniasis. These data indicate that TCRV gamma 1.1+ T cells confer spontaneous immunity against haematopoietic tumours in vivo and link innate resistance to bacterial infections with tissue-specific tumour surveillance by gamma delta+ T cells.

A role for CD4+ T cells in the pathogenesis of skin fibrosis in tight skin mice.

The tight skin (Tsk/+) mouse represents a murine model of heritable fibrosis with some similarities to the skin fibrosis seen in human scleroderma. Tsk/+ animals display alterations in connective tissue in some internal organs. Skin fibrosis can be adoptively transferred to normal recipients with Tsk/+ bone marrow or spleen cells and older Tsk/+ animals develop autoantibodies against topoisomerase suggesting that some of the pathogenesis in the Tsk/+ mouse may be mediated by autoimmunity. To determine the role of T cell subsets in the pathogenesis of fibrotic disease, Tsk/+ mice were bred with CD4- and CD8-deficient (CD4-/- and CD8-/-) mice. Tsk/+ CD4-/- mice showed a marked reduction in skin fibrosis as well as decreased cellularity and only mild collagen disorganization as compared to Tsk/+ CD4+ CD8+ control mice yet did not differ from Tsk controls in the level of serum anti-topoisomerase activity. In contrast, Tsk/+ CD8-/- mice exhibited the same histology in the skin as Tsk/+ controls yet had significantly reduced levels of serum anti-topoisomerase activity. Lung pathology, i.e. emphysema, was unaffected by both the CD4 or CD8 mutations. These data show that only some of the pathological effects of the Tsk mutation are T cell dependent and that different T cell subsets affect different parameters in this multi-organ model of fibrotic disease.