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.

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.

Clonal analysis of F1 hybrid helper T cells. I-A subregion-encoded hybrid determinants restrict the activity of keyhole limpet hemocyanin-specific helper T cells.

Clonal expansion of isolated precursors to helper T cells was induced in limiting dilution cultures of keyhole limpet hemocyanin (KLH)-primed F1 hybrid lymph node cells. Progeny of each isolated precursor was tested for helper activity by transfer to independent cultures with hapten-primed B cells of either parental or F1 hybrid origin. The major histocompatibility complex (MHC) restriction specificity of each F1 hybrid helper T clone was determined. To assess the contribution of I-A and I-E subregion-encoded genes to the expression of these restriction elements, helper T cell cultures derived from F1 hybrids between strains with recombinant H-2 haplotypes were analyzed. Parental and unique F1 hybrid MHC determinants that are encoded entirely within the I-A subregion were found to restrict the activity of KLH-specific helper T cells.

Clonal analysis of F1 hybrid helper T cells restricted to parental or F1 hybrid major histocompatibility determinants.

Antigen-specific major histocompatibility complex (MHC)-restricted helper T cell precursors were induced to proliferate in cultures of keyhole limpet hemocyanin-primed lymph node cells. Clones of F1 hybrid helper T cells were isolated in limiting-dilution cultures. Each positive culture at a limiting-dilution of lymph node cells gave rise to > 10 helper T cells with a single MHC-restricted specificity. This made it possible to independently assay specific helper activity of isolated clones in secondary cultures with B cells of diverse origin. Different clones with helper activity restricted to either parental or unique F1 hybrid MHC determinants were found to occur at approximately equal frequency. The results are discussed in relation to hybrid Ia specificities and dual-complementing MHC-linked Ir genes.

Gamma gene rearrangement and expression in autoreactive helper T cells.

gamma gene rearrangements similar to those described for cytotoxic T cell lines are found in L3T4+, autoreactive, or KLH-specific cloned helper T cell lines. High levels of gamma RNA transcripts were, in addition, detected in four out of five L3T4+, class II MHC-specific, autoreactive T cell clones, and in at least one of three KLH-specific, class II MHC-restricted clones. This contrasts with previously reported (9) expression of gamma RNA in only 1 of 11 antigen-specific helper T cell lines.

Origin and specificity of autoreactive T cells in antigen-induced populations.

We have characterized the major histocompatibility complex (MHC) specificity of autoreactive T cell clones arising from diverse donors after immunization with different antigens. The MHC fine specificity of autoreactive T cells for unique F1 hybrid determinants of BALB.K X BALB.B F1, and for the mutant I-Ab determinants of the B6.C-H-2bm12 (bm 12) strain is similar to that previously described for antigen-specific T cells. We find, furthermore, that the MHC specificity of autoreactive T cell clones selected from primed populations grown in the absence of Con A-stimulated supernatant factors reflects the predominant MHC restriction specificity of T cells specific for the immunogen. Thus, I-E subregion-specific autoreactive T cells are detected at a much higher frequency after immunization with the I-E-restricted antigen, GL (terpolymer of glutamic acid, lysine, and phenylalanine), than with the predominantly I-A-restricted antigen, keyhole limpet hemocyanin (KLH). These experiments strongly suggest that some autoreactive T cells are derived from antigen-stimulated precursors. This result contrasts with that obtained when autoreactive T cells are selected in bulk cultures, or in the presence of exogenous T cell factors. We conclude that, under optimal conditions, most autoreactive T cells are recruited from a relatively stable pool of predominantly I-A-specific precursors. Autoreactive precursors in this pool might themselves derive from previous antigenic stimulation, or be of independent origin.

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.

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.

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)

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.

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.

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.

TTGG-A-L-specific memory B cells induced in low responder strains.

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 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 low and high responder strains. This finding demonstrates that memory formation in a B cell subpopulation represented by TTGG-A--L-specific precursors is independent of carrier-specific, MHC-restricted helper T cells. This conclusion is further supported by the demonstration in an adoptive transfer model that immunization with TTGG-A--L induces equivalent levels of TTGG-A--L-specific memory B cells in T cell-deficient nude mice and their normal heterozygous littermates.

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.

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.