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.

CD4 expression is differentially required for deletion of MLS-1a-reactive T cells.

Clonal deletion of thymocytes expressing potentially self-reactive T cell receptors (TCRs) occurs during thymocyte ontogeny. Mice deficient for CD4 expression provide a unique model system to study the contribution of the CD4 molecule in negative selection of T cells reactive against the major histocompatibility complex class II-associated retroviral self-superantigen, Mls-1a. In the presence of Mls-1a determinants, mature CD8+ T cells expressing V beta 6, 8.1, and 9 were deleted in CD4-deficient mice, thus demonstrating that TCR affinity for Mls-1a is sufficient for deletion and that a signal through CD4 was not required. However, in instances where the TCR affinity for Mls-1a is low, as in the case of V beta 7+ T cells, CD4 expression was required for clonal deletion. These results demonstrate that for Mls-1a-mediated clonal deletion of T cells, the requirement for the accessory or coreceptor function of CD4 depends on the affinity of the TCR.

The induction of experimental autoimmune myocarditis in mice lacking CD4 or CD8 molecules [corrected].

Experimental induction of most autoimmune diseases appears to depend on the activation of CD4+ T helper cells, while CD8+ lymphocytes may have a role in disease progression. To study the role of CD4+ and CD8+ T cell subsets in T cell-dependent autoimmunity, mice lacking CD4 or CD8 molecules after gene targeting were injected with cardiac myosin to induce organ specific autoimmune myocarditis. Mice homozygous for the CD8 mutation (CD8-/-) developed significantly more severe disease as compared to CD4+/-CD8+/- controls. Surprisingly, CD4-/- mice developed autoimmune myocarditis with infiltration of TCR alpha beta +CD4-CD8- T cells in the heart tissue and appearance of autoantibodies. These data demonstrate that the lack of CD4+ or CD8+ T cells has no significant influence on the initiation of autoimmune myocarditis. CD4+ and CD8+ cells regulate disease severity and these results may explain the occurrence of autoimmunity in CD4 immunodeficiencies.

CD45RA and CD45RBhigh expression induced by thymic selection events.

CD45 is a protein tyrosine phosphatase involved in T and B cell signaling. While peripheral T cells switch CD45 isoforms upon activation, events leading to exon switching during T cell development in the thymus have not been determined. The expression of high molecular weight isoforms of CD45 was examined on thymocytes from nontransgenic and T cell receptor (TCR) transgenic mice. All thymocytes from nontransgenic mice were CD45RB+ as assessed by staining with MB23G2, an anti-CD45RB-specific monoclonal antibody. Interestingly, there was a small population (1-3%) of thymocytes that displayed a higher intensity of staining with MB23G2, CD45RBhigh. CD45RBhigh thymocytes were found in all subsets defined by CD4 and CD8 expression and were also present within the TCR-alpha/beta high population. To analyze whether or not CD45 expression correlated with thymic selection events, expression of CD45RBhigh and a second isoform, CD45RA, was examined on thymocytes from H-Y and 2C TCR transgenic mice and found to correlate with positive and negative selection events but did not occur in nonselecting backgrounds. CD45RA and CD45RBhigh upregulation was also not observed in transgenic mice backcrossed into CD8-deficient mice, a scenario in which there is no positive selection of transgene-expressing thymocytes. These data suggest that modulation of CD45 isoform expression may be involved in thymic selection events.

Requirement for tyrosine kinase p56lck for thymic development of transgenic gamma delta T cells.

The Src-related protein tyrosine kinase p56lck is essential for antigen-specific signal transduction and thymic maturation of T cells that have an alpha beta T cell receptor (TCR), presumably by physical association with CD4 or CD8 molecules. To evaluate the requirement for p56lck in the development of T cells that have gamma delta TCRs, which generally do not express CD4 or CD8, p56lck mutant mice were bred with TCR gamma delta transgenic mice. Few peripheral cells that carried the transgenes could be detected in p56lck-/- mice, although 70 percent of thymocytes were transgenic. Development of transgenic gamma delta+ thymocytes was blocked at an early stage, defined by interleukin-2 receptor alpha expression. However, extrathymic development of CD8 alpha alpha+ TCR gamma delta+ intestinal intraepithelial lymphocytes appeared to be normal. Thus, p56lck is crucial for the thymic, but not intestinal, maturation of gamma delta T cells and may function in thymic development independently of CD4 or CD8.

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.

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.

Progress in T cell biology.

We outline recent work in our laboratories on thymus progenitors, lineages within the thymus, interactions between regulatory and effector lymphocytes, splitting the CD4 (T4) T cell subset, and Ir and Is genes. We highlight the possibilities for future research opened up by the demonstration that certain marrow-derived cell lines can repopulate thymic lobes in culture, and also the deep insight into the logical structure of the lymph node provided by our ability to make an exact comparison between two-cell-type and three-cell-type immunoregulatory clusters.

Failure of anti-inflammatory steroids to inhibit prostaglandin release from the hydronephrotic rabbit kidney.

The release of prostaglandins E2, F2 alpha, I2 and thromboxane A2 from isolated perfused normal and hydronephrotic rabbit kidneys was investigated by extraction and radioimmunoassay. In both types of kidneys, basal PG efflux increased with time and was not altered by co-perfusion with dexamethasone or hydrocortisone. Several vasoactive substances at 1 to 4 micrograms (e.g., bradykinin, angiotensin II, substance P, noradrenaline and vasopressin) caused release of additional amounts of prostaglandins. PGE2 and 6-keto PGF1 alpha were the major prostanoids detected, but substantial amounts of PGF2 alpha were also found. Thromboxane A2 was not released from normal kidneys. In hydronephrotic kidneys there was greatly augmented release of prostaglandins E2 and I2, some increases in PGF2 alpha, and the appearance of substantial amounts of thromboxane A2 (measured as immunoreactive TXB2) when the kidneys were challenged with angiotensin, bradykinin and vasopressin, and smaller augmentation of the response to noradrenaline and substance P. There was no evidence that these evoked increases in renal PG output could be inhibited by dexamethasone or hydrocortisone. Some explanations for the failure of steroids to alter prostanoid metabolism from arachidonate in rabbit kidney are discussed, and it is proposed that there are clear exceptions to the concept that steroids inhibit prostaglandin generation in intact tissues.

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.

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.

Maternal transfer of infectious mouse mammary tumor retroviruses does not depend on clonal deletion of superantigen-reactive V beta 14+ T cells.

Female C3H/HeJ mice maternally transmit through their milk an infectious mouse mammary tumor retrovirus (MMTV) which causes clonal deletion of T cell receptor (TcR)V beta 14+ T cells reactive to the retroviral superantigen (SAG). To test whether CD4+ or CD8+ T cells are crucial for intestinal infection and maternal transfer of exogenous retroviruses, newborn mice lacking CD4 or CD8 molecules after gene targetting were raised by surrogate C3H/HeJ mothers. In CD8-/- mice, clonal deletion of TcRV beta 14+ cells reactive to the SAG from this exogenous MMTV occurred with delayed kinetics. Deletion of TcRV beta 14+ cells was not observed in CD4-/- mice up to 12 months after exposure to the retrovirus. In both CD4-/- and CD8-/- mice TcRV beta 5+ and TcRV beta 11+ T cells were deleted in the presence of genomically integrated endogenous MMTV (Mtv), indicating that the lack of SAG-induced clonal deletion was not due to a general defect in these mutant mouse strains. Although TcRV beta 14+ T cells were not deleted in CD4-/- mice, female CD4-/- mice nursed on C3H/HeJ milk maternally transmitted the retrovirus to their offspring, albeit with delayed kinetics. These data demonstrate that CD4+ and CD8+ lymphocytes influence clonal deletion events and that the mechanisms responsible for clonal deletion of SAG-reactive TcRV beta 14+ T cells may be different from mechanisms which allow the mammary tumor virus to enter the mammary gland and complete its infectious cycle.

Tolerance of self induced in thymus organ culture.

F liver protein occurs in serum at low concentration, and therefore induces tolerance of self only in T cells. T cells which mature in cultured thymus lobes in the absence of this protein become reactive towards it but can be prevented from doing so by exposure to the protein while in culture. The threshold of tolerance induction for this soluble antigen is estimated in this way at approximately 1 microgram/ml, which is slightly less than the threshold of response of primed T cells in a proliferation assay. Freshly isolated thymocytes do not display reactivity to self-F protein, indicating that T cells normally become tolerant while still within the thymus.

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.

Characterization of murine thymic stromal-cell lines immortalized by temperature-sensitive simian virus 40 large T or adenovirus 5 E1a.

The heterogeneity of thymic stromal cells is probably related to their role in providing different microenvironments where T cells can develop. We have immortalized thymic stromal elements using recombinant retroviral constructs containing a temperature-sensitive simian virus 40 (SV40tsA58) large-T antigen gene or the adenovirus 5 E1a region linked to the gene coding for resistance to G418. Cell lines containing the thermolabile large T antigen encoded by SV40 proliferate at the permissive temperature of 33 degrees C and arrest growth when transferred to the nonpermissive temperature of 39 degrees C. At the nonpermissive temperature, ts-derived cell lines are shown to alter their phenotype but remain metabolically active, as indicated by the inducible expression of class I and class II MHC antigens. Here we describe the generation of a total of 84 thymic stromal-cell lines, many of which show distinct morphologic, phenotypic, and functional properties consistent with fibroblastoid, epithelial, or monocytoid origins. Several E1a and SV40tsA58-derived cell lines generated exhibit the epithelial characteristic of desmosome formation and, in addition, two of these lines (15.5 and 15.18) form multicellular complexes (rosettes) when incubated with unfractionated thymocytes from syngeneic mice. A single line (14.5) displays very strong nonspecific esterase activity, suggesting it may represent a macrophagelike cell type. We describe the generation of stromal cell lines with different properties, which is consistent with the heterogeneity found in the thymic microenvironment. In addition to documenting this diversity, these cell lines may be useful tools for studying T-cell development in vitro and give access to model systems in which stromal-thymocyte interactions can be examined.

Screening gene expression libraries for epitopes recognized in Mycobacterium leprae by mouse T cells.

Parasite expression libraries have so far been screened with antibodies, DNA probes or T cell clones. Immunity to many parasites, such as Mycobacterium leprae, is largely mediated by T cells, and so the screening of such libraries for T cell epitopes is an important step toward the development of effective vaccines and diagnostic reagents. A new method for screening of lambda gt11 libraries with uncloned T cell populations is presented here, which takes advantage of the fact that the recombinant proteins contain beta-galactosidase as their leader peptide; this allows them to be semipurified by means of anti-beta-galactosidase antibodies coated on the bottom of microtiter plate wells, within which a proliferation assay can then be carried out. Optimum conditions for the assay were determined, using the M. leprae 18-kDa antigen as a test antigen.

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.

Normal B lymphocyte development but impaired T cell maturation in CD45-exon6 protein tyrosine phosphatase-deficient mice.

The transmembrane tyrosine phosphatase CD45 is expressed in multiple isoforms on all nucleated hematopoietic cells, resulting from alternative splicing of variable exons. We generated mice with a mutation in the variable CD45 exon 6, using homologous recombination. In mice homozygous for the CD45-exon6 mutation, B cells and most T cells did not express CD45. Development of B cells appeared normal, although Ig mu-induced proliferation was completely abrogated. Thymocyte maturation was blocked at the transitional stage from immature CD4+CD8+ to mature CD4+ or CD8+ cells, and only a few T cells could be detected in peripheral lymphoid organs. Clonal deletion of superantigen-reactive T cells still occurred. Cytotoxic T cell responses to lymphocytic choriomeningitis virus were absent in CD45-exon6-/- mice. These data imply that CD45 is differentially required for the development and function of B and T lymphocytes.