COVID-19 in patients with lung cancer.

BACKGROUND: Patients with lung cancers may have disproportionately severe coronavirus disease 2019 (COVID-19) outcomes. Understanding the patient-specific and cancer-specific features that impact the severity of COVID-19 may inform optimal cancer care during this pandemic. PATIENTS AND METHODS: We examined consecutive patients with lung cancer and confirmed diagnosis of COVID-19 (n = 102) at a single center from 12 March 2020 to 6 May 2020. Thresholds of severity were defined a priori as hospitalization, intensive care unit/intubation/do not intubate ([ICU/intubation/DNI] a composite metric of severe disease), or death. Recovery was defined as >14 days from COVID-19 test and >3 days since symptom resolution. Human leukocyte antigen (HLA) alleles were inferred from MSK-IMPACT (n = 46) and compared with controls with lung cancer and no known non-COVID-19 (n = 5166). RESULTS: COVID-19 was severe in patients with lung cancer (62% hospitalized, 25% died). Although severe, COVID-19 accounted for a minority of overall lung cancer deaths during the pandemic (11% overall). Determinants of COVID-19 severity were largely patient-specific features, including smoking status and chronic obstructive pulmonary disease [odds ratio for severe COVID-19 2.9, 95% confidence interval 1.07-9.44 comparing the median (23.5 pack-years) to never-smoker and 3.87, 95% confidence interval 1.35-9.68, respectively]. Cancer-specific features, including prior thoracic surgery/radiation and recent systemic therapies did not impact severity. Human leukocyte antigen supertypes were generally similar in mild or severe cases of COVID-19 compared with non-COVID-19 controls. Most patients recovered from COVID-19, including 25% patients initially requiring intubation. Among hospitalized patients, hydroxychloroquine did not improve COVID-19 outcomes. CONCLUSION: COVID-19 is associated with high burden of severity in patients with lung cancer. Patient-specific features, rather than cancer-specific features or treatments, are the greatest determinants of severity.

Anti-CXCL13 antibody can inhibit the formation of gastric lymphoid follicles induced by Helicobacter infection.

Helicobacter suis infects the stomachs of both animals and humans, and can induce gastric mucosa-associated lymphoid tissue (MALT) lymphomas. It is known that CXC chemokine ligand 13 (CXCL13) is highly expressed in the Helicobacter-infected mice and gastric MALT lymphoma patients, but the pathway that links the activation of CXCL13 and the formation of gastric MALT lymphomas remains unclear. In this study, we examined whether CXCL13 neutralization would interfere with the formation of gastric lymphoid follicles including B cells, CD4+T cells, dendritic cells (DCs), and follicular DCs (FDCs) in germinal centers to determine the role of CXCL13 in the formation of B-cell aggregates after H. suis infection. Moreover, the expression of genes associated with the lymphoid follicle formation was also effectively suppressed by anti-CXCL13 antibody treatment. These results suggest that the upregulation of CXCL13 has an important role in the development of gastric MALT lymphomas and highlight the potential of anti-CXCL13 antibody for protection against Helicobacter-induced gastric diseases.

Prior exposure of mice to Fusobacterium nucleatum modulates host response to Porphyromonas gingivalis.

Multiple periodontal pathogens sequentially colonize the subgingival niche during the conversion from gingivitis to destructive periodontal disease. An animal model of sequential immunization with key periodontal pathogens has been developed to determine whether T- and B-lymphocyte effector functions are skewed and fail to protect the host from pathogenic challenge. The present study was performed to evaluate the immunomodulatory effect of exposure to Fusobacterium nucleatum prior to Porphyromonas gingivalis. Group 1 (control) mice were immunized with phosphate-buffered saline, group 2 were immunized with F. nucleatum prior to P. gingivalis and group 3 were immunized with P. gingivalis alone. All the T-cell clones derived from group 2 demonstrated type 2 helper T-cell clone (Th2 subsets), whereas those from group 3 mice demonstrated Th1 subsets. Exposure of mice to F. nucleatum prior to P. gingivalis interfered with the opsonophagocytosis function of sera against P. gingivalis. In adoptive T-cell transfer experiments, in vivo protective capacity of type 2 helper T-cell clones (Th2) from group 2 was significantly lower than type 1 helper T-cell clones (Th1) from group 3 against the lethal dose infection of P. gingivalis. Western blot analysis indicated a different pattern of recognition of P. gingivalis fimbrial proteins between sera from group 2 and group 3. In conclusion, these studies suggest that exposure of a host to F. nucleatum prior to the periodontal pathogen P. gingivalis modulates the host immune responses to P. gingivalis at the humoral, cellular and molecular levels.

Lethality-based selection of recombinant genes in mammalian cells: application to identifying tumor antigens.

Many biological processes result in either cell death or cessation of cell growth. However, plasmid- and retrovirus-based mammalian expression vectors in which it has been possible to construct representative cDNA libraries cannot be readily recovered from cells that are not actively dividing. This has limited the efficiency of selection of recombinant genes that mediate either lytic events or growth arrest. Examples include genes that encode the target antigens of cytotoxic T cells, genes that promote stem-cell differentiation and pro-apoptotic genes. We have successfully constructed representative cDNA libraries in a poxvirus-based vector that can be recovered from cells that have undergone lethality-based selection. This strategy has been applied to selection of a gene that encodes a cytotoxic T-cell target antigen common to several independently derived tumors.

Polarization of Porphyromonas gingivalis-specific helper T-cell subsets by prior immunization with Fusobacterium nucleatum.

Antigen-specific T-cell clones were obtained from mice immunized with Fusobacterium nucleatum ATCC 10953 and/or Porphyromonas gingivalis 381. 10 BALB/c mice per group were immunized with F. nucleatum followed by P. gingivalis, or with P. gingivalis alone by intraperitoneal injection of viable microorganisms. Spleen T cells were isolated and stimulated in vitro with viable P. gingivalis cells to establish P. gingivalis-specific T-cell clones. T-cell phenotypes and cytokine profiles were determined along with T-cell responsiveness to F. nucleatum or P. gingivalis. Serum immunoglobulin G antibody titers to F. nucleatum or P. gingivalis were also determined by enzyme-linked immunosorbent assay. All the T-cell clones derived from mice immunized with F. nucleatum followed by P. gingivalis demonstrated Th2 subsets, while those from mice immunized with P. gingivalis alone demonstrated Th1 subsets based on the flow cytometric analysis and cytokine profiles. All T-cell clones from both groups were cross-reactive to both P. gingivalis and F. nucleatum antigens. Phenotypes of T-cell clones were all positive for CD4. Mean post-immune serum IgG antibody levels to F. nucleatum or P. gingivalis were significantly higher than the pre-immune levels (P < 0.05, P < 0.01, respectively). There were no significant differences in the antibody titers between the two groups. It was concluded that P. gingivalis-specific T cells initially primed by cross-reactive F. nucleatum antigens were polarized to Th2 subset, while T cells stimulated with P. gingivalis alone maintained the profile of Th1 subset.

Construction and characterization of vaccinia direct ligation vectors.

Poxvirus vectors are extensively used as expression vehicles for protein and antigen expression in eukaryotic cells. Customarily, the foreign DNA is introduced into the poxvirus genome by homologous recombination. An alternative method using direct ligation vectors has been used to efficiently construct chimeric genomes in situations not readily amenable for homologous recombination. We describe the construction and characterization of a new set of direct ligation vectors designed to be universally applicable for the generation of chimeric vaccinia genomes. These vectors contain the pair of unique restriction sites NotI and ApaI to eliminate religation of poxvirus arms and fix the orientation of the insert DNA behind strongly expressing constitutive vaccinia promoters. The insertion cassette has been placed at the beginning of the thymidine kinase gene in vaccinia to use drug selection in the isolation of recombinants. These viruses provide a set of universally applicable direct ligation poxvirus cloning vectors, extending the utility of poxvirus vectors for construction and expression of complex libraries.

Peptide binding to mixed isotype Abeta(d)Ealpha(d) class II histocompatibility molecules.

Previous studies have demonstrated that mixed isotype A beta(d) E alpha(d) molecules are expressed in transfected cell lines and that the level of expression is very low in normal B cells from H-2(d) mice. T-cell responses restricted by A beta(d) E alpha(d) are induced in H-2(d) mice immunized with the synthetic peptides YL2 and FL2 or with sperm whale myoglobin, despite the low concentration of mixed isotype molecules expressed on antigen-presenting cells. In the present study, the peptide binding behavior of A beta(d) E alpha(d) was investigated. A peptide from the cytoplasmic domain of invariant chain, I(1-18), was observed to bind with high affinity to purified A beta(d) E alpha(d). Binding was optimal at pH 5, indicating that these molecules prefer to bind peptide in the acidic environment of endosomal compartments similar to other murine class II proteins. YL2 and FL2 bind to A beta(d) E alpha(d) with slightly lower affinity. The selective restriction of YL2- and FL2-specific T cells to mixed isotype molecules was accounted for by the observation that these peptides do not bind to either I-E(d) or I-A(d). By contrast, myoglobin peptides bind to both parental and mixed isotype molecules. None of the A beta(d) E alpha(d)-restricted peptide determinants bind to A beta(d) E alpha(d) with extremely high affinity. Thus it is unlikely that these peptides occupy an unusually high fraction of mixed isotype molecules during antigen presentation in vivo. It is more likely that the presence of a subpopulation of high-affinity T cells capable of being stimulated by very low concentrations of A beta(d) E alpha(d)/peptide complexes is responsible for the unusual A beta(d) E alpha(d)-restricted response observed with some antigens.

Limiting dilution analysis of primary cytotoxic T-cell precursors.

An efficient colorimetric assay has been adapted for limiting dilution analysis of cytotoxic T-cell precursors. Application of this assay in a suitable experimental model of thymic education could be especially useful in identifying factors that shape the CD8 T-cell repertoire. The essential elements of such a model are described here and elsewhere.

Deviation of immune response to Chlamydia psittaci outer membrane protein in lipopolysaccharide-hyporesponsive mice.

The outcome of infection is determined by both the quantity and the quality of an induced immune response. In particular, it has been demonstrated for selected pathogens that induction of TH1 or TH2 type helper T-cell subsets determines whether an immune response gives rise to protective immunity or disease-associated immunopathology. The nature of the antigen and the type of antigen-presenting cells recruited in the induction of a response are critical factors that influence the quality of the immune response. Of particular interest in this respect is the immune response to bacterial particles and the impact of cell wall-associated lipopolysaccharide (LPS) on that response. Nonspecific activation of macrophages and B lymphocytes by LPS could skew the phenotype of activated antigen-presenting cells and selectively alter the immunoglobulin isotypes and helper T-cell subsets that are induced following infection. In an initial attempt to detect immune deviation associated with LPS stimulation, we have compared the immunoglobulin isotypes of antibodies specific for the cysteine-rich outer membrane protein Omp2 induced in normal and LPS-hyporesponsive mice following immunization with Chlamydia psittaci strain guinea pig inclusion conjunctivitis whole elementary bodies. We report that there is a dramatic shift of Omp2-specific antibody from predominantly immunoglobulin G2a (IgG2a) isotype in LPS-hyporesponsive mice to high levels of IgG1 isotype in LPS-responder strains. The dependence of the IgG1 isotype shift on the LPS responder status is linked to the structure of the antigen and its natural processing pathway since LPS-hyporesponsive mice are not, in general, deficient in IgG1 antibody production. In particular, the antibody response to purified recombinant Omp2 is predominantly of the IgG1 isotype even in LPS-hyporesponsive mice.

Dissociation of immune determinants of outer membrane proteins of Chlamydia psittaci strain guinea pig inclusion conjunctivitis.

Chlamydia trachomatis is an important human pathogen. Research to develop a Chlamydia vaccine has focused on the major outer membrane protein (MOMP). Determinants of this protein elicit serovar-specific neutralizing antibodies which are thought to play a critical role in protective immunity. MOMP-specific antibody responses are highly variable in the polymorphic population. Genetic factors which might influence the MOMP-specific immune response are consequently of particular interest. The C. psittaci strain guinea pig inclusion conjunctivitis (GPIC) is a natural pathogen of the guinea pig that causes both ocular and genital tract infections that closely resemble those caused by C. trachomatis in humans. As such, it provides an excellent model for disease. In this report, we explore the influence of major histocompatibility complex-linked genes on the MOMP-specific antibody response in mice immunized with either whole GPIC elementary bodies or recombinant GPIC MOMP. Our results indicate that the MOMP-specific antibody response is major histocompatibility complex linked such that mice of the H-2d haplotype are high responders while mice of the H-2k haplotype are low responders. We demonstrate that MOMP-specific B cells are present in H-2k strains which are, however, deficient in MOMP-specific helper T cells. Although immunization of low-MOMP-responder strains with whole chlamydial elementary bodies induces high levels of immunoglobulin G antibody specific for Omp2, the cysteine-rich outer membrane protein, MOMP-specific B cells are unable to receive help from Omp2-specific T cells. The failure of intermolecular help from Omp2-specific T cells and related observations raise important issues regarding the processing and presentation of chlamydial antigens and the design of optimal subunit vaccines.

Shared T cell-defined antigens on independently derived tumors.

We report that a subset of tumors independently derived from a cloned line of contact-inhibited, non-tumorigenic murine fetal fibroblasts confer cross-protective immunity against each other in vivo. Concordant with the in vivo cross-protection, cytolytic T cell clones from mice immunized with one of these tumor lines specifically lyse the three other lines in the same set but do not cross-react with either the nontumorigenic parental line or another similarly derived tumor line representing a different antigenic profile. This and other recent evidence for shared expression of tumor rejection Ag contrasts with the antigenic diversity previously described for chemical- and radiation-induced tumors. In the interpretation of such data it is essential to distinguish between Ag expressed in association with the transformation process and Ag induced by random mutation of already transformed cells.

Major histocompatibility complex determinants select T-cell receptor alpha chain variable region dominance in a peptide-specific response.

Dominant expression of T-cell receptor (TCR) alpha or beta chain variable region (V alpha or V beta) gene families has been observed in the T-cell response to some conventional peptide antigens. Current models for the interaction of TCR V region elements with different determinants of a major histocompatibility complex (MHC)-peptide complex, the normal TCR ligand, suggest that the TCR V-J junctional region (CDR3, where J is joining) is the primary contact with a peptide epitope and that other TCR V region segments may interact directly with neighboring MHC determinants. This suggests that V alpha or V beta dominance in a specific response can be MHC-selected. In this case, if related peptides bind to an MHC molecule in a similar orientation, they could select for identical V alpha or V beta dominance even if they are noncrossreactive at the level of T-cell activation. We have screened for this possibility by introducing minimal conservative substitutions in a synthetic peptide, YYEELLKYYEELLK, that is presented to T cells in association with an uncommon A beta E alpha d mixed Ia isotype. We report here that the peptide variant FFEELLKFFEELLK is noncrossreactive with YYEELLKYYEELLK but appears to preserve the same MHC binding motif since T-cell responses are restricted to the same mixed A beta E alpha isotype. Although the two peptides are noncrossreactive in either direction, the same members of the V alpha 4 gene family are dominantly expressed in T cells specific for either peptide. We conclude that the similar topography of the two MHC-peptide complexes gives functional significance to a unique A beta E alpha determinant that selects for V alpha 4 dominance.

Specific T-cell response to a Pneumocystis carinii surface glycoprotein (gp120).

Pneumocystis carinii gp120 can elicit a specific T-cell proliferative response in mice after immunization with a gp120 preparation or with a crude P. carinii homogenate. It can also elicit a proliferative response from SCID mice after recovery from natural infection with P. carinii, implicating this glycoprotein as an important antigen in the host's response to P. carinii infection.

Specific T-cell response to a Pneumocystis carinii surface glycoprotein (gp120) after immunization and natural infection.

T cells have been shown to be important in recovery from Pneumocystis carinii pneumonitis, although no specific antigen of P. carinii has been defined as containing T-cell epitopes. P. carinii has an abundant mannosylated surface glycoprotein of approximately 120 kDa (gp120) which induces a prominent host antibody response in experimental animals after exposure to P. carinii in the environment or after recovery from P. carinii pneumonitis. P. carinii gp120 was purified from infected lungs by lectin affinity chromatography. Standard in vitro lymphocyte stimulation assays using purified gp120 and control normal lung preparations were performed on isolated T cells obtained from BALB/c mice after immunization with P. carinii-infected crude lung homogenates or lectin-purified gp120. Lymphocytes from reconstituted severe combined immunodeficient mice which had recovered from naturally acquired P. carinii pneumonitis were also tested. A specific T-cell response was elicited by gp120 after immunization with P. carinii gp120 and after recovery from P. carinii pneumonitis. In addition, the mice developed a strong antibody response to gp120 as ascertained by Western blot (immunoblot). These data suggest that gp120 may be important in the recognition of P. carinii by T cells.

Imprint of thymic selection on autoreactive repertoires.

We have focussed on the differences in origin and physiological properties of two classes of self-reactive T cells. Autoreactive T cells described in many laboratories are activated in the course of normal immune responses to foreign antigen. These T cells can be shown under well-defined conditions to be the direct progeny of antigen-stimulated precursors. This, together with evidence that their activation requirements can be distinguished from those of antigen-specific, MHC-restricted T cells, leads us to suggest that they represent a particular physiological state that recapitulates the conditions of thymic selection and is induced in many antigen-specific, MHC-restricted peripheral T cells as a result of normal antigen-dependent activation. Although it appears that the associated physiological properties can be stable in some in vitro maintained lines, it is possible that this is normally a transient state in vivo. Available evidence concerning the specificity of these T cells indicates only that they can be activated in the absence of any identifiable foreign antigen by class II MHC-syngeneic but not MHC-allogeneic stimulators. We have suggested that such T cells are specific for the same elements, possibly an association of MHC and other self-peptides (Singer et al. 1987), that are the basis for positive selection in the thymus. The properties of these autoreactive T cells need to be distinguished from those of T cells associated with autoimmune pathology. It is presumed that autoimmune T cells are directly activated in a resting state by specific self-peptides. Our interest in distinguishing these self-reactive T-cell populations has focussed on different predictions concerning the diversity of their associated self-reactive repertoires. The relative complexity of the immune repertoire expressed in autoreactive T cells expanded by positive selection and restimulated in the course of normal antigen-specific immune responses should be considerably greater than that of autoimmune T cells constrained by negative selection and a narrow window of escape from self-tolerance. We were greatly hindered in our initial efforts in this analysis by the considerable effort required to characterize any specific immune repertoire. A published technique employing poly(A) tailing (Frohman et al. 1988) did not work efficiently in our hands, although others (Loh et al. 1989) have apparently had some success. We describe above an alternative approach, linker-facilitated PCR, which we have employed for efficient repertoire analysis. Using this method we have been able to identify dominant utilization of the Va4 family in T cells specific for the synthetic peptide YYEELLKYYEELLK.(ABSTRACT TRUNCATED AT 400 WORDS)

Autoreactive T-cell response to resting or activated B cells.

Cloned autoreactive T-cell lines and hybridomas have been selected in many laboratories. A number of observations have suggested that activation of Ia-positive stimulators may be required for optimal induction of an autoreactive response. We have examined the ability of small resting B cells fractionated by centrifugal elutriation to stimulate the proliferative response of seven cloned autoreactive T-cell lines. Of these, six were efficiently stimulated by resting B cells. One I-Ed-specific Th2-type T-cell clone failed to be stimulated by resting B cells. This clone did, however, respond to this same cell fraction following lipopolysaccharide (LPS) activation. An independent I-E-specific Th2 clone was stimulated by resting B cells. It appears, therefore, that a requirement for activated stimulators is not a general property of either autoreactive T cells or the Th2 helper T-cell subset.

Requirements for stimulation of autoreactive T cells by thymic stroma.

Thymic stromal cells are more efficient than similarly treated spleen cells for Ag presentation to Ag-specific, MHC-restricted T cell lines. Thymic stromal cells fail, however, to stimulate proliferation of autoreactive T cell lines. This failure to stimulate autoreactive T cells does not appear to be due to tolerance induction because thymic stroma does not interfere with subsequent stimulation by spleen cells. Moreover, the ability of thymic stromal cells to stimulate autoreactive T cells can be restored by addition of exogenous IL-1. This demonstrates that the specific self-determinants recognized by autoreactive T cells can be expressed on thymic stromal cells. Failure of stimulation by thymic stromal cells in the absence of exogenous IL-1 may reflect a difference in the physiologic requirements for activation of autoreactive T cells as compared to Ag-specific, MHC-restricted T cells.

Different T cell requirements for specific memory induction in normal and xid B cells.

The immune response to TTGG-A--L, a defined-sequence, branched-chain polypeptide, is regulated by MHC-linked Ir genes. TTGG-A--L specific B cells can be demonstrated in both normal and immune-defective low responder strains by activation to specific antibody secretion after immunization with TTGGAA-F gamma G, a conjugate of the hexapeptide TTGGAA and the immunogenic carrier fowl gamma-globulin. It is shown that immunization with TTGG-A--L induces specific memory B cells with equal efficiency in normal low and high responder strains but not in immune-defective low responder strains. We conclude that memory induction in xid B cells in contrast to normal B cells is dependent on MHC-restricted, carrier-specific helper T cells. Other observations also suggest a more stringent requirement for MHC-restricted, carrier-specific helper T cells in the induction of TTGGAA-specific antibody secretion by xid as compared to normal B cells. Both normal and immune-defective H-2k/b hybrids between the mutant CBA/N strain and TTGG-A--L high responder BALB.B are responders to TTGG-A--L. In contrast, normal but not immune-defective H-2k/d hybrids with responder BALB/c are responders to TTGG-A--L. This identifies H-2d as a TTGG-A--L high responder haplotype for normal B cells but a low responder haplotype for xid B cells, whereas H-2b is a high responder haplotype for both normal and xid B cells. This must reflect a quantitative or qualitative difference in Ir gene-mediated cellular interactions required for induction of antibody secretion in normal and xid B cells.